U.S. patent application number 11/048774 was filed with the patent office on 2006-03-02 for pancreas-derived plasminogen activator inhibitor.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Reiner L. Gentz, Jian Ni, Steven M. Ruben, Y. Eric Shi.
Application Number | 20060045878 11/048774 |
Document ID | / |
Family ID | 26697980 |
Filed Date | 2006-03-02 |
United States Patent
Application |
20060045878 |
Kind Code |
A1 |
Ni; Jian ; et al. |
March 2, 2006 |
Pancreas-derived plasminogen activator inhibitor
Abstract
The present invention relates to a novel member of the
plasminogen activator inhibitor protein family. In particular,
isolated nucleic acid molecules are provided encoding the
pancreas-derived plasminogen activator inhibitor protein.
Pancreas-derived plasminogen activator inhibitor polypeptides are
also provided as are vectors, host cells and recombinant methods
for producing the same. The invention further relates to methods
for treating physiologic and pathologic disease conditions and
diagnostic methods for detecting pathologic disorders.
Inventors: |
Ni; Jian; (Germantown,
MD) ; Gentz; Reiner L.; (Belo Horizonte-Mg, BR)
; Ruben; Steven M.; (Brookeville, MD) ; Shi; Y.
Eric; (Roslyn Heights, NY) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
26697980 |
Appl. No.: |
11/048774 |
Filed: |
February 3, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10628395 |
Jul 29, 2003 |
6893870 |
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11048774 |
Feb 3, 2005 |
|
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|
09902684 |
Jul 12, 2001 |
6649738 |
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|
10628395 |
Jul 29, 2003 |
|
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|
09026408 |
Feb 19, 1998 |
6303338 |
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09902684 |
Jul 12, 2001 |
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08934011 |
Aug 15, 1997 |
|
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09026408 |
Feb 19, 1998 |
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60024056 |
Aug 16, 1996 |
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Current U.S.
Class: |
424/133.1 ;
435/320.1; 435/329; 435/69.1; 530/387.3; 536/23.53 |
Current CPC
Class: |
C07K 14/8132 20130101;
A61K 48/00 20130101; Y02A 50/30 20180101; Y02A 50/463 20180101;
C07K 2319/00 20130101; C07K 14/81 20130101; A61K 38/00
20130101 |
Class at
Publication: |
424/133.1 ;
435/069.1; 435/320.1; 435/329; 530/387.3; 536/023.53 |
International
Class: |
C07H 21/04 20060101
C07H021/04; A61K 39/395 20060101 A61K039/395; C07K 16/44 20060101
C07K016/44; C12N 5/06 20060101 C12N005/06 |
Claims
1. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide comprising amino acids from about -14 to
about 378 in SEQ ID NO:2; (b) a nucleotide sequence encoding a
polypeptide comprising amino acids from about -13 to about 378 in
SEQ ID NO:2; (c) a nucleotide sequence encoding a polypeptide
comprising amino acids from about 1 to about 378 in SEQ ID NO:2;
(d) a nucleotide sequence encoding a polypeptide comprising amino
acids from about -18 to about 387 in SEQ ID NO:13; (e) a nucleotide
sequence encoding a polypeptide comprising amino acids from about
-17 to about 387 in SEQ ID NO:13; (f) a nucleotide sequence
encoding a polypeptide comprising amino acids from about 1 to about
387 in SEQ ID NO:13; (g) a nucleotide sequence encoding a
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATTC.TM. Deposit No. 97657; (h) a nucleotide
sequence encoding the mature PAPAI polypeptide having the amino
acid sequence encoded by the cDNA clone contained in ATTC.TM.
Deposit No. 97657; and (i) a nucleotide sequence complementary to
any of the nucleotide sequences in (a), (b), (c), (d), (e), (f),
(g), or (h).
2. An isolated nucleic acid molecule comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), (d), (e), (f), (g), (h), or
(i) of claim 1 wherein said polynucleotide which hybridizes does
not hybridize under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence consisting of only A
residues or of only T residues.
3. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a PAPAI polypeptide having an amino acid sequence in (a), (b),
(c), (d), (e), (f), or (g) of claim 1.
4. The isolated nucleic acid molecule of claim 3, which encodes an
epitope-bearing portion of a PAPAI polypeptide selected from the
group consisting of: a polypeptide comprising amino acid residues
from about 6 to about 16 in SEQ ID NO:2; a polypeptide comprising
amino acid residues from about 31 to about 36 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about 46 to about
76 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 111 to about 121 in SEQ ID NO:2; a polypeptide
comprising amino acid residues from about 146 to about 161 in SEQ
ID NO:2; a polypeptide comprising amino acid residues from about
206 to about 211 in SEQ ID NO:2; a polypeptide comprising amino
acid residues from about 236 to about 246 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about 306 to about
316 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 361 to about 366 in SEQ ID NO:2; a polypeptide
comprising amino acid residues from about 2 to about 12 in SEQ ID
NO:13; a polypeptide comprising amino acid residues from about 27
to about 32 in SEQ ID NO:13; a polypeptide comprising amino acid
residues from about 42 to about 72 in SEQ ID NO:13; a polypeptide
comprising amino acid residues from about 107 to about 117 in SEQ
ID NO:13; a polypeptide comprising amino acid residues from about
142 to about 157 in SEQ ID NO:13; a polypeptide comprising amino
acid residues from about 202 to about 207 in SEQ ID NO:13; a
polypeptide comprising amino acid residues from about 232 to about
242 in SEQ ID NO:13; a polypeptide comprising amino acid residues
from about 302 to about 312 in SEQ ID NO:13; and a polypeptide
comprising amino acid residues from about 357 to about 362 in SEQ
ID NO:13.
5. A method for making a recombinant vector comprising inserting an
isolated nucleic acid molecule of claim 1 into a vector.
6. A recombinant vector produced by the method of claim 5.
7. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 6 into a host cell.
8. A recombinant host cell produced by the method of claim 7.
9. A recombinant method for producing a PAPAI polypeptide,
comprising culturing the recombinant host cell of claim 8 under
conditions such that said polypeptide is expressed and recovering
said polypeptide.
10. An isolated PAPAI polypeptide having an amino acid sequence at
least 95% identical to a sequence selected from the group
consisting of: (a) amino acids from about -14 to about 378 in SEQ
ID NO:2; (b) amino acids from about -13 to about 378 in SEQ ID
NO:2; (c) amino acids from about 1 to about 378 in SEQ ID NO:2; (d)
amino acids from about -18 to about 387 in SEQ ID NO:13; (e) amino
acids from about -17 to about 387 in SEQ ID NO:13; (f) amino acids
from about 1 to about 387 in SEQ ID NO:13; (g) the amino acid
sequence of the PAPAI polypeptide having the amino acid sequence
encoded by the cDNA clone contained in ATTC.TM. Deposit No. 97657;
(h) the amino acid sequence of the mature PAPAI polypeptide having
the amino acid sequence encoded by the cDNA clone contained in
ATTC.TM. Deposit No. 97657; and (i) the amino acid sequence of an
epitope-bearing portion of any one of the polypeptides of (a), (b),
(c), (d), (e), (f), (g), or (h).
11. The isolated polypeptide of claim 10, which is produced or
contained in a recombinant host cell.
12. The isolated polypeptide of claim 10, wherein said recombinant
host cell is mammalian.
13. An isolated antibody that binds specifically to a plasminogen
activator inhibitor polypeptide of claim 10.
14. A method for treatment of an individual in need of an increased
level of pancreas-derived plasminogen activator inhibitor activity
comprising administering to said individual a composition
comprising an isolated polypeptide of claim 10.
15. A method for treatment of an individual in need of an increased
level of pancreas-derived plasminogen activator inhibitor activity
comprising administering to said individual a composition
comprising a recombinant vector of claim 6.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/628,395, filed Jul. 29, 2003, which is a divisional of U.S.
application Ser. No. 09/902,684, filed Jul. 12, 2001, which is a
divisional of U.S. application Ser. No. 09/026,408, filed Feb. 19,
1998, which is herein incorporated by reference; said U.S.
application Ser. No. 09/026,408 is a continuation-in-part of U.S.
application Ser. No. 08/934,011, filed Aug. 15, 1997, which is
herein incorporated by reference; said U.S. application Ser. No.
08/934,011 claims benefit under 35 U.S.C. .sctn. 119(e) of U.S.
Provisional Application No. 60/024,056, filed Aug. 16, 1996, which
is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a novel member of the
serine protease inhibitor (serpin) superfamily of proteins, in
particular the plasminogen activator inhibitor (PAI) protein
family. More specifically, isolated nucleic acid molecules are
provided encoding the pancreas-derived plasminogen activator
inhibitor (PAPAI) protein. Plasminogen activator inhibitor
polypeptides are also provided. The present invention further
relates to methods for treating physiologic and pathologic disease
conditions and diagnostic methods for detecting pathologic
disorders.
[0004] 2. Related Art
[0005] The mammalian serine protease inhibitors (serpins) are a
superfamily of single chain proteins that contain a conserved
structure of approximately 370 amino acids and generally range
between 40 and 60 kDa in molecular mass. .alpha..sub.1-Antitrypsin
(also known as .alpha..sub.1-proteinase inhibitor) is a
characteristic member of the serpin family in that it is a single
chain glycoprotein of nearly 400 amino acid residues that functions
by forming a tight 1:1 complex with its cognate protease,
neutrophil (leucocyte) elastase, which subsequently slowly
dissociates to yield active enzyme and inactive cleaved inhibitor
(Carrell, R. W. et al., Cold Spring Harbor Symposia on Quantitative
Biology 52:527-535 (1987)). The reactive center of the serpins is
typically formed by an X-Ser that acts as a substrate for the
target serine protease: .alpha..sub.1-antitrypsin has a Met-Ser
reactive center with the methionine residue providing a putative
cleavage site for neutrophil elastase.
[0006] The majority of serpins function as protease inhibitors and
so are involved in regulation of several proteinase-activated
physiological processes, such as blood coagulation, fibrinolysis,
complement activation, extracellular matrix turnover, cell
migration and prohormone activation (Potempa, J. et al., J. Biol.
Chem. 269:15957-19560 (1994)). As noted, serpins inhibit
proteolytic events by forming a 1:1 stoichiometric complex with the
active site of their cognate proteinases, which is resistant to
denaturants (Cohen, A. B. et al., Biochemistry 17:392-400 (1987).
The serpins include, but are not limited to,
.alpha..sub.1-antitrypsin (.alpha..sub.1-proteinase inhibitor),
antithrombin III, plasminogen activator inhibitor 1 (PAI-1),
plasminogen activator inhibitor 2 (PAI-2),
.alpha..sub.1-antichymotrypsin, and .alpha..sub.2-antiplasmin
(Huber, R. and Carrell, R. W., Biochemistry 28:8951-8966
(1989).
[0007] The plasminogen activator system is responsible for the
degradation of intravascular blood clots, and also contributes to
extracellular proteolysis in a wide variety of physiological
processes of normal development and pathological processes in the
etiology of diseases such as tumor invasion and metastasis
(Andreasen, P.-A., et al., Int. J. Cancer 72(1):1-22 (1997);
Schmitt, M., et al., Thromb. Haemost. 78(1):285-296 (1997)).
Plasmin, a trypsin-like protease, is generated from its precursor
plasminogen by the action of plasminogen activators, of which there
are two types: tissue-type plasminogen activator (also known as
tissue plasminogen activator) and urokinase. Plasmin degrades
fibrin and several extracellular matrix and adhesion proteins and
activates procollagenases.
[0008] Plasminogen activation is a highly regulated process.
Precise, coordinated, spatial and temporal regulation is afforded
by the interaction of a variety of mechanisms. These mechanisms
include (1) inhibition by specific plasmin and plasminogen
activator inhibitors; (2) binding of plasminogen, plasminogen
activators, and inhibitors to fibrin, extracellular matrix
proteins, and specific cell surface receptors; (3) release of
tissue plasminogen activator and inhibitors from intracellular
storage granules; (4) regulation of gene expression of plasminogen
activators and inhibitors; (5) an autocrine feedback loop whereby
plasmin-mediated activation of latent forms of growth factors
regulates the expression of activators and inhibitors; and (6)
clearance of free and inhibitor-bound activators via receptors
(Bachmann, F. et al., Fibrinolysis, in: Thrombosis Haemostasis
1987, Verstraete, M. et al., eds., Leuven University Press (1987);
Dano, K. et al., Adv. Cancer Res. 44:139 (1985); Pollanen, J. et
al., Adv. Cancer Res. 57:273 (1991); Vassalli, J. D. et al., J.
Clin. Invest. 88:1067 (1991); Carmeliet, P. et al., Thromb.
Haemost. 74:429 (1995); Andreasen, P. A. et al., Mol. Cell.
Endocrinol. 68:1 (1990); Loskutoff, D. J., Fibrinolysis 5:197
(1991); Keski-Oja, J. et al., Semin. Thromb. Hemost. 17:231 (1991);
Blasi, F., BioEssays 15:105 (1993); Andreasen, P. A. et al., FEBS
Lett. 338:239 (1994); Bu, G. et al., Blood 83:3427 (1994); and
Camani, C. et al., Int. J. Hematol. 60:97 (1994)).
[0009] Strong clinical and experimental evidences have strongly
suggested an important and apparently causal role for the
tumor-associated urokinase-type PA (u-PA) and the receptor u-PAR in
cancer invasion and metastasis (Andreasen, P.-A., et al., Int. J.
Cancer 72(1):1-22 (1997); Schmitt, M., et al., Thromb. Haemost.
78(1):285-296 (1997); Duggan, C., et al., Br. J. Cancer
76(5):622-627 (1997)). Consistent with its role in cancer
metastasis, overexpression and unrestrained activity of u-PA has
been shown to be a prognostic marker in many different types of
human cancer (Schmitt, M., et al., Fibrinolysis 6(Suppl. 4):3-26
(1992); Schmitt, M., et al., J Obstet. Gynaecol. 21:151-165 (1995);
Brunner, N., et al., Cancer Treat. Res. 71:299-309 (1994); Kuhn,
W., et al., Gynecol. Oncol. 55:401409 (1994); Ganesh, S., et al.,
Cancer Res. 54:4065-4071 (1994); Nekarda, H., et al., Cancer Res.
54:2900-2907 (1994); Duffy, M.-J., J. Clin. Cancer Res. 2:613-618
(1996)). The down-regulation of u-PA may occur at the levels of
transcriptional regulation of the genes and through interaction
with specific endogenous inhibitors such as plasminogen activator
inhibitor (PAI).
[0010] Only two plasminogen activator inhibitors are known. These
are plasminogen activator inhibitor 1 and 2 (PAI-1 and PAI-2,
respectively). PAI-1 and PAI-2 regulate mitogenesis, adhesion of
myeloid cells, fusion of myoblasts, and migration of endothelial
cells (Fazioli, F. et al., Trends Pharm. Sci. 15:25-29 (1995)).
Indeed, PAI-1 and PAI-2 are involved in many physiological and
pathological processes, including normal pregnancy, preeclampsia,
intrauterine growth retardation, wound healing, tumor cell invasion
and metastasis, inflammation and arthritis, inflammatory bowel
disease, appendicitis, complications from systemic lupus
erythematosus, ovulation and prostatic involution and osteonecrosis
(Kruithof, E. K. O. et al., Blood 86:4007 (1995)).
[0011] Both PAI-1 and PAI-2 have been shown to inhibit
extracellular matrix degradation in vitro (Cajot, J.-F., et al.,
Proc. Natl. Acad. Sci. USA 87:6939-6943 (1990); Baker, M.-S., et
al., Cancer Res. 50:4676-4684 (1990)). These results suggest that
the inhibitory activity of PAIs might be important in inhibiting
tumor malignant progression leading to metastasis. In fact,
administration of a recombinant PAI-2 to mice decreases tumor
growth (Astedt, B., et al., Fibrinol. 9:175-177 (1995)), whereas
overexpression of either PAI-1 or PAI-2 inhibits tumor metastasis
(Muller, B., et al., Proc. Natl. Acad. Sci. USA 92:205-209 (1995);
Soff, G.-A., et al., J. Clin. Invest. 96:2593-2600 (1995)).
[0012] In breast cancer, it has been demonstrated extensively that
uPA and PAI-1 are statistically independent, strong prognostic
factors for disease free and overall survival, i.e., high tumor
levels are associated with a poor prognosis and are conductive to
tumor cell spread and metastasis (Bmmner, N., et al., Cancer Treat.
Res. 71:299-309 (1994); Duffy, M., et al., Cancer 62:531-533
(1988); Duggan, C., et al., Int. J. Cancer 61:597-600 (1995);
Schmitt, M., et al., Br. J. Cancer 76(3):306-311 (1997)).
Immunohistochemical staining has detected PAI-1 expression at
stromal fibroblasts surrounding tumor nodules or at tumor margins
(Bianchi, E., et al., Int. J. Cancer 60:597-603 (1995)). The
production of PAI-1 by the tumor stroma may represent a host
defensive response to the excessive proteolysis. In contrast to
PAI-1, high level PAI-2 expression may be a favorable prognostic
marker in breast cancer (Schmitt, M., et al., Thromb. Haemost.
78(1):285-296 (1997); Duggan, C., et al., Br. J. Cancer
76(5):622-627 (1997)). In breast carcinomas with high uPA values,
PAI-2 was associated with a prolonged relapse-free survival,
metastasis-free survival, and overall survival (Bouchet, C., et
al., Br. J. Cancer 69:398-405 (1994)). In relation to the
clinicopathological findings, an inverse correlation between PAI-2
mRNA expression and lymph node metastasis was demonstrated in
breast cancers (Sumiyoshi, K., et al., S. Int. J. Cancer 50:345-348
(1992)). In this study, the expression of uPA and PAI-1 was
significantly correlated with negative expression of PAI-2; and a
low level of PAI-2 expression was significantly associated with
lymph node involvement (Sumiyoshi, K., et al., Int. J. Cancer
50:345-348 (1992)). PAI-2 expression is detected predominantly in
malignant mammary epithelial cells of primary carcinomas but is
also present in stromal cells (Andreasen, P.-A., et al., Int. J.
Cancer 72(1): 1-22 (1997)). These results indicate that PAI-2 may
play a critical role in inhibition of extracellular matrix
degradation mediated by plasminogen activator during tumor cell
invasion and metastasis.
[0013] In view of the wide range of roles that plasminogen
activator inhibitors play in physiologic and pathologic processes,
there is a continuing need for the isolation and characterization
of novel plasminogen activator inhibitors.
SUMMARY OF THE INVENTION
[0014] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding the pancreas-derived
plasminogen activator inhibitor (PAPAI) polypeptide having the
amino acid sequence is shown in FIGS. 1A-1B (SEQ ID NO:2), FIGS.
4A-4B (SEQ ID NO:13) or the amino acid sequence encoded by the cDNA
clone deposited in a bacterial host as ATTC.TM. Deposit Number
97657 on Jul. 12, 1996. The nucleotide sequence determined by
sequencing the deposited PAPAI clone, which is shown in FIGS.
4A-4B, contains an open reading frame encoding a polypeptide of 405
amino acid residues, including an initiation codon at positions
67-69, with a leader sequence of about 18 amino acid residues, and
a deduced molecular weight of about 46 kDa. The amino acid sequence
of the mature PAPAI protein is shown in SEQ ID NO:13 (amino acid
residues from about 1 to about 387 in SEQ ID NO:13). Another
sequence of a PAPAI clone which is shown in FIGS. 1A-1B (SEQ ID
NO:1), contains an open reading frame encoding a polypeptide of 392
amino acid residues, including an initiation codon at positions
67-69, with a leader sequence of about 14 amino acid residues, and
a deduced molecular weight of about 44.5 kDa. The amino acid
sequence of this mature PAPAI protein is shown in SEQ ID NO:2
(amino acid residues from about 1 to about 378 in SEQ ID NO:2).
[0015] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding the PAPAI polypeptide having the
complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide
sequence encoding the PAPAI polypeptide having the complete amino
acid sequence in SEQ ID NO:2 but minus the N-terminal methionine
residue; (c) a nucleotide sequence encoding the mature PAPAI
polypeptide having the amino acid sequence at positions 1-378 in
SEQ ID NO:2; (d) a nucleotide sequence encoding the PAPAI
polypeptide having the complete amino acid sequence in SEQ ID
NO:13; (e) a nucleotide sequence encoding the PAPAI polypeptide
having the complete amino acid sequence in SEQ ID NO:13, but minus
the N-terminal methionine residue; (f) a nucleotide sequence
encoding the mature PAPAI polypeptide having the amino acid
sequence at positions 1-387 in SEQ ID NO:13; (g) a nucleotide
sequence encoding the PAPAI polypeptide having the complete amino
acid sequence encoded by the cDNA clone contained in ATTC.TM.
Deposit No. 97657; (h) a nucleotide sequence encoding the mature
PAPAI polypeptide having the amino acid sequence encoded by the
cDNA clone contained in ATTC.TM. Deposit No. 97657; and (i) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), or (h) above.
[0016] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above,
or a polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c), (d), (e), (f),
(g), (h), or (i) above. This polynucleotide which hybridizes does
not hybridize under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence consisting of only A
residues or of only T residues. An additional nucleic acid
embodiment of the invention relates to an isolated nucleic acid
molecule comprising a polynucleotide which encodes the amino acid
sequence of an epitope-bearing portion of a PAPAI polypeptide
having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g),
(h), or (i) above.
[0017] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of PAPAI polypeptides or peptides by
recombinant techniques.
[0018] The invention further provides an isolated PAPAl polypeptide
having amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of the PAPAI polypeptide having the
complete 392 amino acid sequence, including the leader sequence
shown in SEQ ID NO:2; (b) the amino acid sequence of the PAPAI
polypeptide having the complete 392 amino acid sequence, including
the leader sequence shown in SEQ ID NO:2, but minus the N-terminal
methionine residue; (c) the amino acid sequence of the mature PAPAI
polypeptide (without the leader) having the amino acid sequence at
positions 1-378 in SEQ ID NO:2; (d) the amino acid sequence of the
PAPAI polypeptide having the complete 405 amino acid sequence,
including the leader sequence shown in SEQ ID NO:13; (e) the amino
acid sequence of the PAPAI polypeptide having the complete 405
amino acid sequence, including the leader sequence shown in SEQ ID
NO:13, but minus the N-terminal methionine residue; (f) the amino
acid sequence of the mature PAPAI polypeptide (without the leaser)
having the amino acid sequence at positions 1-387 in SEQ ID NO:13;
(g) the amino acid sequence of the PAPAI polypeptide having the
complete amino acid sequence, including the leader, encoded by the
cDNA clone contained in ATTC.TM. Deposit No. 97657; and (h) the
amino acid sequence of the mature PAPAI polypeptide having the
amino acid sequence encoded by the cDNA clone contained in ATTC.TM.
Deposit No. 97657. The polypeptides of the present invention also
include polypeptides having an amino acid sequence at least 95%
identical, more preferably at least 96%, 97%, 98% or 99% identical
to those above.
[0019] An additional embodiment of this aspect of the invention
relates to a peptide or polypeptide which has the amino acid
sequence of an epitope-bearing portion of a PAPAI polypeptide
having an amino acid sequence described in (a), (b), (c), (d), (e),
(f), (g), or (h) above. Peptides or polypeptides having the amino
acid sequence of an epitope-bearing portion of a PAPAI polypeptide
of the invention include portions of such polypeptides with at
least six or seven, preferably at least nine, and more preferably
at least about 30 amino acids to about 50 amino acids, although
epitope-bearing polypeptides of any length up to and including the
entire amino acid sequence of a polypeptide of the invention
described above also are included in the invention. In another
embodiment, the invention provides an isolated antibody that binds
specifically to a PAPAI polypeptide having an amino acid sequence
described in (a), (b), (c), (d), (e), (f), (g), or (h) above.
[0020] For a number of pathologic disorders, such as tumor invasion
and metastasis, significant alterations (increases or decreases) in
level of PAPAI gene expression can be detected in a sample of
tissue or bodily fluid. Increased or decreased levels of PAPAI gene
expression can be measured, in such a sample, relative to a
"standard" PAPAI gene expression level, i.e., the PAPAI expression
level in a tissue or bodily fluid from an individual not having the
disorder. Thus, the present invention provides a diagnostic method
useful during diagnosis of such disorders, which involves assaying
the expression level of the gene encoding the PAPAI protein in
tissue or bodily fluid from an individual and comparing the gene
expression level with a standard PAPAI gene expression level,
whereby an increase or decrease in the gene expression level over
the standard is indicative of a pathologic disorder, such as tumor
invasion and metastasis, hemorrhage in liver disease, and
preeclampsia.
[0021] The PAPAl protein inhibits the plasminogen activator system
when administered to an individual. The plasminogen activator
system is responsible for the degradation of intravascular blood
clots, while also contributing to extracellular proteolysis in a
wide variety of physiological processes (e.g. wound healing, cell
migration, tissue remodeling, angiogenesis, trophoblast
implantation, ovulation and fetal development) and pathological
processes (e.g. tumor invasion and metastasis, intrauterine growth
retardation, preeclampsia, and acute and chronic inflammation).
Thus, by the invention, methods are provided for inhibiting the
plasminogen activator system, which involve administering an
inhibitory amount of PAPAI either alone or together with one or
more plasminogen activator inhibitors, such as PAI-1 and PAI-2.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIGS. 1A-1B show the nucleotide (SEQ ID NO:1) and deduced
amino acid (SEQ ID NO:2) sequences of pancreas-derived plasminogen
activator inhibitor (PAPAI) protein. The protein has a leader
sequence of about 14 amino acid residues (underlined) and a deduced
molecular weight of about 44.5 kDa. The predicted amino acid
sequence of the rnature PAPAI protein is also shown in FIGS. 1A-1B
(SEQ ID NO:2).
[0023] FIGS. 2A-2C show the regions of similarity between the amino
acid sequences of the PAPAI protein (HPASD50P protein) and human
plasminogen activator inhibitor 1 (PAI-1) (SEQ ID NO:3) and human
plasminogen activator inhibitor 2 (PAI-2) (SEQ ID NO:4).
[0024] FIG. 3 shows an analysis of the predicted alpha, beta, turn,
and coil regions, and the predicted hydrophilicity, amphipathic
nature, flexible regions, antigenic index, and surface probability
plot of the of the polypeptide of FIGS. 1A-1B (SEQ ID NO: 2) and
FIGS. 4A-4B (SEQ ID NO:13). In the "Antigenic Index--Jameson-Wolf"
graph, amino acid residues about 20 to about 30, about 45 to about
50, about 60 to about 90, about 125 to about 135, about 160 to
about 175, about 220 to about 225, about 250 to about 260, about
320 to about 330, and about 375 to about 380 in FIGS. 1A-1B and
FIGS. 4A-4B correspond to the highly antigenic regions of the PAPAI
protein. These highly antigenic fragments in FIGS. 1A-1B correspond
to the following fragments, respectively, in SEQ ID NO:2: amino
acid residues about 6 to about 16, about 31 to about 36, about 46
to about 76, about 111 to about 121, about 146 to about 161, about
206 to about 211, about 236 to about 246, about 306 to about 316,
and about 361 to about 366. These highly antigenic fragments in
FIGS. 4A-4B correspond to the following fragments, respectively, in
SEQ ID NO:13: amino acid residues about 2 to about 12, about 27 to
about 32, about 42 to about 72, about 107 to about 117, about 142
to about 157, about 202 to about 207, about 232 to about 242, about
302 to about 312, and about 357 to about 362.
[0025] FIGS. 4A-4B show the nucleotide (SEQ ID NO:12) and deduced
amino acid (SEQ ID NO:13) sequences of PAPAI, which was determined
by sequencing the cDNA clone deposited as ATTC.TM. Deposit No.
97657. The protein has a leader sequence of about 18 amino acid
residues (underlined) and a deduced molecular weight of about 46
kDa.
[0026] FIG. 5 shows a schematic representation of the pHE4-5
expression vector (SEQ ID NO:14) and the subcloned PAPAI cDNA
coding sequence. The locations of the kanamycin resistance marker
gene, the PAPAI coding sequence, the oriC sequence, and the lacIq
coding sequence are indicated.
[0027] FIG. 6 shows the nucleotide sequence of the regulatory
elements of the pHE promoter (SEQ ID NO:15). The two lac operator
sequences, the Shine-Delgamo sequence (S/D), and the terminal
HindIII and NdeI restriction sites (italicized) are indicated.
DETAILED DESCRIPTION
[0028] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a PAPAI polypeptide,
having the amino acid sequence shown in FIGS. 4A-4B (SEQ ID NO:13),
which was determined by sequencing a cloned cDNA, or in FIGS. 1A-1B
(SEQ ID NO:2). The PAPAI protein of the present invention shares
sequence homology with human plasminogen activator inhibitor 1
(PAI-1) (SEQ ID NO:3) and human plasminogen activator 2 (PAI-2)(SEQ
ID NO:4). The nucleotide sequence shown in FIGS. 4A-4B (SEQ ID
NO:13) was obtained by sequencing the HPASD50P clone, which was
deposited on Jul. 12, 1996 at the American Type Culture Collection,
Patent Depository, 10801 University Boulevard, Manassas, Va.,
20110-2209, and given accession number 97657.
[0029] Accordingly, in one embodiment of the present invention,
isolated nucleic acid molecules are provided which encode the PAPAI
protein. PAPAI is a novel member of the plasminogen activator
inhibitor subfamily.
Nucleic Acid Molecules
[0030] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were
predicted by translation of a DNA sequence determined as above.
Therefore, as is known in the art for any DNA sequence determined
by this automated approach, any nucleotide sequence determined
herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more
typically at least about 95% to at least about 99.9% identical to
the actual nucleotide sequence of the sequenced DNA molecule. The
actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in
the art. As is also known in the art, a single insertion or
deletion in a determined nucleotide sequence compared to the actual
sequence will cause a frame shift in translation of the nucleotide
sequence such that the predicted amino acid sequence encoded by a
determined nucleotide sequence will be completely different from
the amino acid sequence actually encoded by the sequenced DNA
molecule, beginning at the point of such an insertion or
deletion.
[0031] Unless otherwise indicated, each "nucleotide sequence" set
forth herein is presented as a sequence of deoxyribonucleotides
(abbreviated A, G, C and T). However, by "nucleotide sequence" of a
nucleic acid molecule or polynucleotide is intended, for a DNA
molecule or polynucleotide, a sequence of deoxyribonucleotides, and
for an RNA molecule or polynucleotide, the corresponding sequence
of ribonucleotides (A, G, C and U), where each thymidine
deoxyribonucleotide (T) in the specified deoxyribonucleotide
sequence is replaced by the ribonucleotide uridine (U). For
instance, reference to an RNA molecule having the sequence of SEQ
ID NO:1 set forth using deoxyribonucleotide abbreviations is
intended to indicate an RNA molecule having a sequence in which
each deoxyribonucleotide A, G or C of SEQ ID NO:1 has been replaced
by the corresponding ribonucleotide A, G or C, and each
deoxyribonucleotide T has been replaced by a ribonucleotide U.
[0032] Using the information provided herein, such as the
nucleotide sequence in SEQ ID NO:1 or SEQ ID NO:12, a nucleic acid
molecule of the present invention encoding a PAPAI polypeptide may
be obtained using standard cloning and screening procedures, such
as those for cloning cDNAs using mRNA as starting material.
Illustrative of the invention, the nucleic acid molecules described
in SEQ ID NO:1 and SEQ ID NO:12 were discovered in a cDNA library
derived from human pancreatic tissue. The determined nucleotide
sequence of the PAPAI cDNA of SEQ ID NO:1 contains an open reading
frame encoding a protein of 392 amino acid residues, with an
initiation codon at positions 67-69 of the nucleotide sequence in
SEQ ID NO:1, a predicted leader sequence of about 14 amino acid
residues, and a deduced molecular weight of about 44.5 kDa. The
amino acid sequence of the predicted mature PAPAI is shown in SEQ
ID NO:2 from amino acid residue 1 to residue 378. The PAPAI protein
shown in SEQ ID NO:2 is about 67% and 68% identical to PAI-1 (SEQ
ID NO:3) and PAI-2 (SEQ ID NO:4), respectively (FIGS. 2A-2C). The
determined nucleotide sequence of the PAPAI cDNA of SEQ ID NO:12
contains an open reading frame encoding a protein of 405 amino acid
residues, with an initiation codon at positions 67-69 of the
nucleotide sequence in SEQ ID NO:12, a predicted leader sequence of
about 18 amino acid residues, and a deduced molecular weight of
about 46 kDa. The amino acid sequence of the predicted mature PAPAI
is shown in SEQ ID NO:13 from amino acid residue 1 to residue 387.
The PAPAI protein shown in SEQ ID NO:12 was determined by
sequencing the deposited clone. As one of ordinary skill would
appreciate, due to the possibilities of sequencing errors discussed
above, as well as the variability of cleavage sites for leaders in
different known proteins, the actual PAPAI polypeptide encoded by
the deposited cDNA comprises about 405 amino acids, but may be
anywhere in the range of 385-425 amino acids; and the actual leader
sequence of this protein is about 18 amino acids, but may be
anywhere in the range of about 10 to about 26 amino acids.
[0033] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0034] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0035] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) with
an initiation codon at positions 67-69 of the nucleotide sequence
shown in SEQ ID NO:1; DNA molecules comprising the coding sequence
for the mature PAPAI protein shown in FIGS. 1A-1B (last 378 amino
acids) (SEQ ID NO:2); DNA molecules comprising an ORF with an
initiation codon at positions 67-69 of the nucleotide sequence
shown in SEQ ID NO:12; DNA molecules comprising the coding sequence
for the mature PAPAI protein shown in FIGS. 4A-4B (last 387 amino
acids); and DNA molecules which comprise a sequence substantially
different from those described above but which, due to the
degeneracy of the genetic code, still encode the PAPAI protein. Of
course, the genetic code is well known in the art. Thus, it would
be routine for one skilled in the art to generate the degenerate
variants described above.
[0036] In addition, the present inventors have identified nucleic
acid molecules having nucleotide sequences related to extensive
portions of SEQ ID NO:1 which have been determined from the
following related cDNA clones: HPASD50R (SEQ ID NO:10) and
HBXFM84RA (SEQ ID NO:11).
[0037] In another aspect, the invention provides isolated nucleic
acid molecules encoding the PAPAI polypeptide having an amino acid
sequence encoded by the cDNA clone contained in the plasmid
deposited as ATTC.TM. Deposit No. 97657 on Jul. 12, 1996. In a
further embodiment, nucleic acid molecules are provided encoding
the mature PAPAI polypeptide or full-length PAPAI polypeptide
lacking the N-terminal methionine residue. The invention further
provides an isolated nucleic acid molecule having the nucleotide
sequence shown in SEQ ID NO:1 or the nucleotide sequence of the
PAPAI cDNA contained in the above-described deposited clone, or a
nucleic acid molecule having a sequence complementary to one of the
above sequences. Such isolated molecules, particularly DNA
molecules, are useful as probes for gene mapping, by in situ
hybridization with chromosomes, and for detecting expression of the
PAPAI gene in human tissue, for instance, by Northern blot
analysis.
[0038] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in ATTC.TM.
Deposit 97657. By "stringent hybridization conditions" is intended
overnight incubation at 42.degree. C. in a solution comprising: 50%
formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times. Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm
DNA, followed by washing the filters in 0.1.times.SSC at about
65.degree. C. By a polynucleotide which hybridizes to a "portion"
of a polynucleotide is intended a polynucleotide (either DNA or
RNA) hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 nt of the
reference polynucleotide. These are useful as diagnostic probes and
primers as discussed above and in more detail below.
[0039] Of course, polynucleotides hybridizing to a larger portion
of the reference polynucleotide (e.g., the deposited cDNA clone),
for instance, a portion 50, 100, 150, 200, 250, 300, 350, 400, 450,
500, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, or 1100 nt in
length, or even to the entire length of the reference
polynucleotide, are also useful as probes according to the present
invention, as are polynucleotides corresponding to most, if not
all, of the nucleotide sequence of the deposited cDNA or the
nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:12. By a
portion of a polynucleotide of "at least 20 nt in length," for
example, is intended 20 or more contiguous nucleotides from the
nucleotide sequence of the reference polynucleotide (e.g., the
deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1
or SEQ ID NO:12). As indicated, such portions are useful
diagnostically either as a probe according to conventional DNA
hybridization techniques or as primers for amplification of a
target sequence by the polymerase chain reaction (PCR), as
described, for instance, in Molecular Cloning, A Laboratory Manual,
2nd. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis,
T., (1989), Cold Spring Harbor Laboratory Press, the entire
disclosure of which is hereby incorporated herein by reference.
[0040] Since a PAPAI cDNA clone has been deposited and its
determined nucleotide sequence is provided in SEQ ID NO:1 and SEQ
ID NO:12, generating polynucleotides which hybridize to a portion
of the PAPAI cDNA molecule would be routine to the skilled artisan.
For example, restriction endonuclease cleavage or shearing by
sonication of the PAPAI cDNA clone could easily be used to generate
DNA portions of various sizes which are polynucleotides that
hybridize to a portion of the PAPAI cDNA molecule. Alternatively,
the hybridizing polynucleotides of the present invention could be
generated synthetically according to known techniques. Of course, a
polynucleotide which hybridizes only to a poly A sequence (such as
the 3' terminal poly(A) tract of the PAPAI cDNA shown in FIGS.
1A-1B (SEQ ID NO:1) or FIGS. 4A-4B (SEQ ID NO:12)), or to a
complementary stretch of T (or U) resides, would not be included in
a polynucleotide of the invention used to hybridize to a portion of
a nucleic acid of the invention, since such a polynucleotide would
hybridize to any nucleic acid molecule containing a poly (A)
stretch or the complement thereof (e.g., practically any
double-stranded cDNA clone).
[0041] The invention further provides isolated nucleic acid
molecules comprising a polynucleotide encoding an epitope-bearing
portion of the PAPAI protein. In particular, isolated nucleic acid
molecules of the present invention include nucleic acid molecules
encoding: a polypeptide comprising amino acid residues from about 6
to about 16 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about 31 to about 36 in SEQ ID NO:2; a polypeptide
comprising amino acid residues from about 46 to about 76 in SEQ ID
NO:2; a polypeptide comprising amino acid residues from about 111
to about 121 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about 146 to about 161 in SEQ ID NO:2; a polypeptide
comprising amino acid residues from about 206 to about 211 in SEQ
ID NO:2; a polypeptide comprising amino acid residues from about
236 to about 246 in SEQ ID NO:2; a polypeptide comprising amino
acid residues from about 306 to about 316 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about 361 to about
366 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 2 to about 12 in SEQ ID NO:13; a polypeptide comprising
amino acid residues from about 27 to about 32 in SEQ ID NO:13; a
polypeptide comprising amino acid residues from about 42 to about
72 in SEQ ID NO:13; a polypeptide comprising amino acid residues
from about 107 to about 117 in SEQ ID NO:13; a polypeptide
comprising amino acid residues from about 142 to about 157 in SEQ
ID NO:13; a polypeptide comprising amino acid residues from about
202 to about 207 in SEQ ID NO:13; a polypeptide comprising amino
acid residues from about 232 to about 242 in SEQ ID NO:13; a
polypeptide comprising amino acid residues from about 302 to about
312 in SEQ ID NO:13; and a polypeptide comprising amino acid
residues from about 357 to about 362 in SEQ ID NO:13. Methods for
generating such epitope-bearing portions of PAPAI are described in
detail below.
[0042] As indicated, nucleic acid molecules of the present
invention which encode a PAPAI polypeptide may include, but are not
limited to those encoding the amino acid sequence of the mature
polypeptide, by itself; the coding sequence for the mature
polypeptide and additional sequences, such as those encoding the
about 14 amino acid leader or secretory sequence, such as a pre-,
or pro- or prepro-protein sequence; the coding sequence of the
mature polypeptide, with or without the aforementioned additional
coding sequences, together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities. Thus, the sequence encoding the polypeptide may be
fused to a marker sequence, such as a sequence encoding a peptide
which facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (Qiagen, Inc.), among others, many of
which are commercially available. As described in Gentz et al.,
Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin protein, which has been described by Wilson et al.,
Cell 37: 767 (1984).
[0043] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the PAPAI protein. Variants may
occur naturally, such as a natural allelic variant. By an "allelic
variant" is intended one of several alternate forms of a gene
occupying a given locus on a chromosome of an organism. Genes II,
Lewin, B., ed., John Wiley & Sons, New York (1985).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0044] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the PAPAI protein or portions thereof. Also
especially preferred in this regard are conservative substitutions.
Most highly preferred are nucleic acid molecules encoding the
mature PAPAI protein having the amino acid sequence shown in FIGS.
1A-1B (SEQ ID NO:2) or FIGS. 4A-4B (SEQ ID NO:13) or the mature
PAPAI amino acid sequence encoded by the deposited cDNA clone.
[0045] Further embodiments of the invention include isolated
nucleic acid molecules comprising a polynucleotide having a
nucleotide sequence at least 95% identical, and more preferably at
least 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence
encoding the polypeptide having the amino acid sequence in SEQ ID
NO:2; (b) a nucleotide sequence encoding the polypeptide having the
amino acid sequence in SEQ ID NO:2, but lacking the N-temminal
methionine residue; (c) a nucleotide sequence encoding the
polypeptide having the amino acid sequence at positions from about
1 to about 378 in SEQ ID NO:2; (d) a nucleotide sequence encoding
the polypeptide having the amino acid sequence in SEQ ID NO:13; (e)
a nucleotide sequence encoding the polypeptide having the amino
acid sequence in SEQ ID NO:13, but lacking the N-terminal
methionine residue; (f) a nucleotide sequence encoding the
polypeptide having the amino acid sequence at positions from about
1 to about 387 in SEQ ID NO:13; (g) a nucleotide sequence encoding
the polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATTC.TM. Deposit No. 97657; (h) a nucleotide
sequence encoded by the cDNA clone contained in ATTC.TM. Deposit
No. 97657; or (i) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), or
(h).
[0046] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a PAPAI polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the PAPAI polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0047] As a practical matter, whether any particular nucleic acid
molecule is at least 95%, 96%, 97%, 98% or 99% identical to, for
instance, the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID
NO:12 or to the nucleotides sequence of the deposited cDNA clone
can be determined conventionally using known computer programs such
as the Bestfit program (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, Madison, Wis. 53711. Bestfit uses the
local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2: 482-489 (1981), to find the best segment of homology
between two sequences. When using Bestfit or any other sequence
alignment program to determine whether a particular sequence is,
for instance, 95% identical to a reference sequence according to
the present invention, the parameters are set, of course, such that
the percentage of identity is calculated over the full length of
the reference nucleotide sequence and that gaps in homology of up
to 5% of the total number of nucleotides in the reference sequence
are allowed.
[0048] The present application is directed to nucleic acid
molecules at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:12 or to
the nucleic acid sequence of the deposited cDNA, irrespective of
whether they encode a polypeptide having PAPAI activity. This is
because even where a particular nucleic acid molecule does not
encode a polypeptide having PAPAI activity, one of skill in the art
would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having PAPAI activity
include, inter alia, (1) isolating the PAPAI gene or allelic
variants thereof in a cDNA library; (2) in situ hybridization
(e.g., "FISH") to metaphase chromosomal spreads to provide precise
chromosomal location of the PAPAI gene, as described in Verma et
al., Human Chromosomes: A Manual of Basic Techniques, Pergamon
Press, New York (1988); and Northern Blot analysis for detecting
PAPAI mRNA expression in specific tissues.
[0049] Preferred, however, are nucleic acid molecules having
sequences at least 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:12 or to
the nucleic acid sequence of the deposited cDNA which do, in fact,
encode a polypeptide having PAPAI protein activity. By "a
polypeptide having PAPAI activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the PAPAI protein of the invention (either the
full-length protein or, preferably, the mature protein), as
measured in a particular biological assay.
[0050] Assays of plasminogen activator activity are well-known to
those in the art. These assays can be used to measure plasminogen
activator activity of partially purified or purified native or
recombinant protein. For example, an .sup.125I fibrin lysis assay
can be used (Lyon, P. B. et al., The Prostate 27:179-186 (1995);
Unkeless, J. C. et al., J. Exp. Med 137:85-126 (1973)).
[0051] In this assay, .sup.125I fibrinogen is placed into 96-well,
flat bottom culture plates at a concentration of 10 .mu.g/cm.sup.2
in a volume of 10-30 .mu.l. Dried plates are exposed to 100 .mu.l
volume of RPMI medium containing 10% fetal bovine serum. Excess
thrombin in the serum results in fibrinogen conversion to fibrin
with a total trypsinizable radioactivity of approximately 60,000
counts per minute per well. Into each test well is placed 1-5 IU of
tissue plasminogen activator (Sigma, St. Louis, Mo.).
Alternatively, if cells are used in place of tissue plasminogen
activator, 1.times.10.sup.5 cells, washed twice with
phosphate-buffered saline, are placed into each test well.
[0052] Into each test well is placed 1 .mu.g of human plasminogen
and 1 to 10 .mu.g of the partially purified or purified native or
recombinant test protein, in 5-50 .mu.l of phosphate buffered
saline.
[0053] Control wells receive an equal volume (5-50 .mu.l) of
phosphate buffered saline. Each sample is assayed in duplicate or
triplicate, with background values of radioactive release
determined from the duplicate sample wells without plasminogen.
Additional control wells include media alone, media plus
plasminogen, media plus plasminogen and plasminogen activator, and
wells containing up to 200 .mu.l of 0.25% bovine trypsin to
determine the maximal releasable radioactivity.
[0054] At intervals following plating, the medium is removed and
radioactivity is measured by gamma counting. Plasminogen-dependent
fibrinolysis is defined as the difference in supernatant
radioactivity between plasminogen-containing and plasminogen-free
wells. Plasminogen activator-dependent fibrinolysis is expressed as
a percentage of the maximal releasable radioactivity per well.
Maximal releasable radioactivity is determined by averaging the
total trypsinizable radioactivity in three fibrin-coated wells. It
will be apparent to one of ordinary skill in the art that the
amounts of reactants and reactant conditions may have to be
modified in order to practice the assay.
[0055] PAPAI inhibits plasminogen activators such as urokinase and
tissue plasminogen activator. Thus, "a polypeptide having PAPAI
protein activity" includes polypeptides that exhibit the
plasminogen activator inhibiting activity, in the above-described
assay and in a dose-dependent manner. Although the degree of
dose-dependent activity need not be identical to that of the PAPAI
protein, preferably, "a polypeptide having PAPAI protein activity"
will exhibit substantially similar dose-dependence in a given
activity as compared to the PAPAI protein (i.e., the candidate
polypeptide will exhibit greater activity or not more than about
tenfold less and, preferably, not more than about twofold less
activity relative to the reference PAPAI protein).
[0056] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence
of the deposited cDNA or the nucleic acid sequence shown in SEQ ID
NO:1 or SEQ ID NO:12 will encode a polypeptide "having PAPAI
protein activity." In fact, since degenerate variants of these
nucleotide sequences all encode the same polypeptide, this will be
clear to the skilled artisan even without performing the above
described comparison assay. It will be further recognized in the
art that, for such nucleic acid molecules that are not degenerate
variants, a reasonable number will also encode a polypeptide having
PAPAI protein activity. This is because the skilled artisan is
fully aware of amino acid substitutions that are either less likely
or not likely to significantly effect protein function (e.g.,
replacing one aliphatic amino acid with a second aliphatic amino
acid).
[0057] For example, guidance concerning how to make phenotypically
silent amino acid substitutions is provided in Bowie, J. U. et al.,
"Deciphering the Message in Protein Sequences: Tolerance to Amino
Acid Substitutions," Science 247:1306-1310 (1990), wherein the
authors indicate that there are two main approaches for studying
the tolerance of an amino acid sequence to change. The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality. As the authors state, these
studies have revealed that proteins are surprisingly tolerant of
amino acid substitutions. The authors further indicate which amino
acid changes are likely to be permissive at a certain position of
the protein. For example, most buried amino acid residues require
nonpolar side chains, whereas few features of surface side chains
are generally conserved. Other such phenotypically silent
substitutions are described in Bowie, J. U. et al., supra, and the
references cited therein.
Vectors and Host Cells
[0058] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of PAPAI polypeptides or fragments thereof by
recombinant techniques.
[0059] Recombinant constructs may be introduced into host cells
using well known techniques such as infection, transduction,
transfection, transvection, electroporation and transformation. The
vector may be, for example, a phage, plasmid, viral or retroviral
vector. Retroviral vectors may be replication competent or
replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells.
[0060] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0061] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0062] In certain preferred embodiments in this regard, the vectors
provide for specific expression, which may be inducible and/or cell
type-specific. Particularly preferred among such vectors are those
inducible by environmental factors that are easy to manipulate,
such as temperature and nutrient additives.
[0063] Expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors
derived from bacterial plasmids, bacteriophage, yeast episomes,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses, vaccinia viruses, adenoviruses, fowl pox viruses,
pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof, such as cosmids and phagemids.
[0064] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp and tac promoters, the SV40 early and late promoters
and promoters of retroviral LTRs, to name a few. Other suitable
promoters will be known to the skilled artisan. The expression
constructs will further contain sites for transcription initiation,
termination and, in the transcribed region, a ribosome binding site
for translation. The coding portion of the mature transcripts
expressed by the constructs will preferably include a translation
initiating at the beginning and a termination codon (UAA, UGA or
UAG) appropriately positioned at the end of the polypeptide to be
translated.
[0065] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline or ampicillin in resistance genes for culturing in E.
coli and other bacteria. Representative examples of appropriate
hosts include, but are not limited to, bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells; fungal cells,
such as yeast cells; insect cells such as Drosophila S2 and
Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes
melanoma cells; and plant cells. Appropriate culture mediums and
conditions for the above-described host cells are known in the
art.
[0066] Among vectors preferred for use in bacteria include pQE70,
pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,
available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRIT5 available from Pharmacia. Among preferred eukaryotic vectors
are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;
and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other
suitable vectors will be readily apparent to the skilled
artisan.
[0067] Among known bacterial promoters suitable for use in the
present invention include the E. coli lacI and lacZ promoters, the
T3 and T7 promoters, the gpt promoter, the lambda PR and PL
promoters and the trp promoter. Suitable eukaryotic promoters
include the CMV immediate early promoter, the HSV thymidine kinase
promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and
metallothionein promoters, such as the mouse metallothionein-I
promoter.
[0068] In addition to the use of expression vectors in the practice
of the present invention, the present invention further includes
novel expression vectors comprising operator and promoter elements
operatively linked to nucleotide sequences encoding a protein of
interest. One example of such a vector is pHE4-5 which is described
in detail below.
[0069] As summarized in FIGS. 5 and 6, components of the pHE4-5
vector (SEQ ID NO:14) include: 1) a neomycinphosphotransferase gene
as a selection marker, 2) an E. coli origin of replication, 3) a T5
phage promoter sequence, 4) two lac operator sequences, 5) a
Shine-Delgarno sequence, 6) the lactose operon repressor gene
(lacIq). The origin of replication (oriC) is derived from pUC19
(LTI, Gaithersburg, Md.). The promoter sequence and operator
sequences were made synthetically. Synthetic production of nucleic
acid sequences is well known in the art. CLONTECH.TM. 95/96
Catalog, pages 215-216, CLONTECH.TM., 1020 East Meadow Circle, Palo
Alto, Calif. 94303. A nucleotide sequence encoding PAPAI (SEQ ID
NO:2 or 13), is operatively linked to the promoter and operator by
inserting the nucleotide sequence between the NdeI and Asp718 sites
of the pHE4-5 vector.
[0070] As noted above, the pHE4-5 vector contains a lacIq gene.
Laclq is an allele of the lacI gene which confers tight regulation
of the lac operator. Amann, E. et al., Gene 69.301-315 (1988);
Stark, M., Gene 51:255-267 (1987). The lacIq gene encodes a
repressor protein which binds to lac operator sequences and blocks
transcription of down-stream (i.e., 3') sequences. However, the
lacIq gene product dissociates from the lac operator in the
presence of either lactose or certain lactose analogs, e.g.,
isopropyl B-D-thiogalactopyranoside (IPTG). PAPAI thus is not
produced in appreciable quantities in uninduced host cells
containing the pHE4-5 vector. Induction of these host cells by the
addition of an agent such as IPTG, however, results in the
expression of the PAPAI coding sequence.
[0071] The promoter/operator sequences of the pHE4-5 vector (SEQ ID
NO:15) comprise a T5 phage promoter and two lac operator sequences.
One operator is located 5' to the transcriptional start site and
the other is located 3' to the same site. These operators, when
present in combination with the lacIq gene product, confer tight
repression of down-stream sequences in the absence of a lac operon
inducer, e.g., IPTG. Expression of operatively linked sequences
located down-stream from the lac operators may be induced by the
addition of a lac operon inducer, such as IPTG. Binding of a lac
inducer to the lacIq proteins results in their release from the lac
operator sequences and the initiation of transcription of
operatively linked sequences. Lac operon regulation of gene
expression is reviewed in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH
CLINICAL CORRELATIONS, 4th Edition (1997), pages 802-807.
[0072] The pHE4 series of vectors contain all of the components of
the pHE4-5 vector except for the PAPAI coding sequence. Features of
the pHE4 vectors include optimized synthetic T5 phage promoter, lac
operator, and Shine-Delgamo sequences. Further, these sequences are
also optimally spaced so that expression of an inserted gene may be
tightly regulated and high level of expression occurs upon
induction.
[0073] Among known bacterial promoters suitable for use in the
production of proteins of the present invention include the E. coli
lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter,
the lambda PR and PL promoters and the trp promoter. Suitable
eukaryotic promoters include the CMV immediate early promoter, the
HSV thymidine kinase promoter, the early and late SV40 promoters,
the promoters of retroviral LTRs, such as those of the Rous Sarcoma
Virus (RSV), and metallothionein promoters, such as the mouse
metallothionein-I promoter.
[0074] The pHE4-5 vector also contains a Shine-Delgamo sequence 5'
to the AUG initiation codon. Shine-Delgamo sequences are short
sequences generally located about 10 nucleotides up-stream (i.e.,
5') from the AUG initiation codon. These sequences essentially
direct prokaryotic ribosomes to the AUG initiation codon.
[0075] Thus, the present invention is also directed to expression
vector useful for the production of the proteins of the present
invention. This aspect of the invention is exemplified by the
pHE4-5 vector (SEQ ID NO:14).
[0076] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0077] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp that
act to increase transcriptional activity of a promoter in a given
host cell-type. Examples of enhancers include the SV40 enhancer,
which is located on the late side of the replication origin at bp
100 to 270, the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and
adenovirus enhancers.
[0078] For secretion of the translated protein into the lumen of
the endoplasmic reticulum, into the periplasmic space or into the
extracellular environment, appropriate secretion signals may be
incorporated into the expressed polypeptide. The signals may be
endogenous to the polypeptide or they may be heterologous
signals.
[0079] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. Thus, for
instance, a region of additional amino acids, particularly charged
amino acids, may be added to the N-termiinus of the polypeptide to
improve stability and persistence in the host cell, during
purification, or during subsequent handling and storage. Also,
peptide moieties may be added to the polypeptide to facilitate
purification.
[0080] The PAPAI protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include naturally purified products, products of chemical synthetic
procedures, and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial,
yeast, higher plant, insect and mammalian cells. Depending upon the
host employed in a recombinant production procedure, the
polypeptides of the present invention may be glycosylated or may be
non-glycosylated. In addition, polypeptides of the invention may
also include an initial modified methionine residue, in some cases
as a result of host-mediated processes.
PAPAI Polypeptides and Fragments
[0081] The invention further provides an isolated PAPAI polypeptide
having the amino acid sequence encoded by the deposited cDNA, or
the amino acid sequence in SEQ ID NO:2 or SEQ ID NO:13, or a
peptide or polypeptide comprising a portion of the above
polypeptides. The terms "peptide" and "oligopeptide" are considered
synonymous (as is commonly recognized) and each term can be used
interchangeably as the context requires to indicate a chain of at
least to amino acids coupled by peptidyl linkages. The word
"polypeptide" is used herein for chains containing more than ten
amino acid residues. All oligopeptide and polypeptide formulas or
sequences herein are written from left to right and in the
direction from amino terminus to carboxy terminus.
[0082] It will be recognized in the art that some amino acid
sequences of the PAPAI polypeptide can be varied without
significant effect of the structure or function of the protein. If
such differences in sequence are contemplated, it should be
remembered that there will be critical areas on the protein which
determine activity. In general, it is possible to replace residues
which form the tertiary structure, provided that residues
performing a similar function are used. In other instances, the
type of residue may be completely unimportant if the alteration
occurs at a non-critical region of the protein.
[0083] Thus, the invention further includes variations of the PAPAI
polypeptide which show substantial PAPAI polypeptide activity or
which include regions of PAPAI protein such as the protein portions
discussed below. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions (for example,
substituting one hydrophilic residue for another, but not strongly
hydrophilic for strongly hydrophobic as a rule). Small changes or
such "neutral" amino acid substitutions will generally have little
effect on activity.
[0084] Typically seen as conservative substitutions are the
replacements, one for another, among the aliphatic amino acids Ala,
Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,
exchange of the acidic residues Asp and Glu, substitution between
the amide residues Asn and Gln, exchange of the basic residues Lys
and Arg and replacements among the aromatic residues Phe, Tyr.
[0085] As indicated in detail above, further guidance concerning
which amino acid changes are likely to be phenotypically silent
(i.e., are not likely to have a significant deleterious effect on a
function) can be found in Bowie, J. U., et al., "Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid
Substitutions," Science 247:1306-1310 (1990).
[0086] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2 or SEQ ID NO:13, or that encoded by the deposited
cDNA, may be (i) one in which one or more of the amino acid
residues are substituted with a conserved or non-conserved amino
acid residue (preferably a conserved amino acid residue) and such
substituted amino acid residue may or may not be one encoded by the
genetic code, or (ii) one in which one or more of the amino acid
residues includes a substituent group, or (iii) one in which the
mature polypeptide is fused with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as an IgG Fc fusion
region peptide or leader or secretory sequence or a sequence which
is employed for purification of the mature polypeptide or a
proprotein sequence. Such fragments, derivatives and analogs are
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0087] Of particular interest are substitutions of charged amino
acids with another charged amino acid and with neutral or
negatively charged amino acids. The latter results in proteins with
reduced positive charge to improve the characteristics of the PAPAI
protein. The prevention of aggregation is highly desirable.
Aggregation of proteins not only results in a loss of activity but
can also be problematic when preparing pharmaceutical formulations,
because they can be immunogenic. (Pinckard et al., Clin Exp.
Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36:838-845
(1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems
10:307-377 (1993)).
[0088] As indicated, changes are preferably of a minor nature, such
as conservative amino acid substitutions that do not significantly
affect the folding or activity of the protein (see Table 1).
TABLE-US-00001 TABLE 1 Conservative Amino Acid Substitutions.
Aromatic Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine
Isoleucine Valine Polar Glutamine Asparagine Basic Arginine Lysine
Histidine Acidic Aspartic Acid Glutamic Acid Small Alanine Senne
Threonine Methionine Glycine
[0089] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of substitutions
for any given PAPAI polypeptide will not be more than 50, 40, 30,
20, 10, 5, or 3.
[0090] Amino acids in the PAPAI protein of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as plasminogen activator
inhibition. Sites that are critical for plasminogen activator
inhibition can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos
et al. Science 255:306-312 (1992)).
[0091] The polypeptides of the present invention are preferably
provided in an isolated form. By "isolated polypeptide" is intended
a polypeptide removed from its native environment. Thus, a
polypeptide produced and/or contained within a recombinant host
cell is considered isolated for purposes of the present invention.
Also intended as an "isolated polypeptide" are polypeptides that
have been purified, partially or substantially, from a recombinant
host cell or a native source. For example, a recombinantly produced
version of the PAPAI polypeptide can be substantially purified by
the one-step method described in Smith and Johnson, Gene 67:31-40
(1988).
[0092] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the PAPAI polypeptide
can be substantially purified by the one-step method described in
Smith and Johnson, Gene 67:31-40 (1988).
[0093] The polypeptides of the present invention include the
polypeptide encoded by the deposited cDNA including the leader; the
mature polypeptide encoded by the deposited the cDNA minus the
leader (i.e., the mature protein); a polypeptide comprising amino
acids about -14 to about 378 in SEQ ID NO:2; a polypeptide
comprising amino acids about -13 to about 378 in SEQ ID NO:2; a
polypeptide comprising amino acids about 1 to about 378 in SEQ ID
NO:2; a polypeptide comprising amino acids about -18 to about 387
in SEQ ID NO:13; a polypeptide comprising amino acids about -17 to
about 387 in SEQ ID NO:13; a polypeptide comprising amino acids
about 1 to about 387 in SEQ ID NO:13; as well as polypeptides at
least 95% identical, more preferably at least 96%, 97%, 98% or 99%
identical to the polypeptide encoded by the deposited cDNA, to the
polypeptide of SEQ ID NO:2 or SEQ ID NO:13, and also include
portions of such polypeptides with at least 30 amino acids and more
preferably at least 50 amino acids.
[0094] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
PAPAI polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the PAPAI
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0095] As a practical matter, whether any particular polypeptide is
at least 95%, 96% 97%, 98% or 99% identical to, for instance, the
amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:13 or to the
amino acid sequence encoded by deposited cDNA clone can be
determined conventionally using known computer programs such the
Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711. When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0096] As described in detail below, the polypeptides of the
present invention can be used to raise polyclonal and monoclonal
antibodies, which are useful in diagnostic assays for detecting
PAPAI protein expression as described below or as agonists and
antagonists capable of enhancing or inhibiting PAPAI protein
function. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" PAPAI protein binding proteins which
are also candidate agonist and antagonist according to the present
invention. The yeast two hybrid system is described in Fields and
Song, Nature 340:245-246 (1989).
[0097] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. These immunogenic epitopes are believed to be confined
to a few loci on the molecule. On the other hand, a region of a
protein molecule to which an antibody can bind is defined as an
"antigenic epitope." The number of immunogenic epitopes of a
protein generally is less than the number of antigenic epitopes.
See, for instance, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998-4002 (1983).
[0098] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983) Antibodies that react with predetermined sites on proteins.
Science 219:660-666. Peptides capable of eliciting protein-reactive
sera are frequently represented in the primary sequence of a
protein, can be characterized by a set of simple chemical rules,
and are confined neither to immunodominant regions of intact
proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl
terminals. Peptides that are extremely hydrophobic and those of six
or fewer residues generally are ineffective at inducing antibodies
that bind to the mimicked protein; longer, soluble peptides,
especially those containing proline residues, usually are
effective. Sutcliffe et al., supra, at 661. For instance, 18 of 20
peptides designed according to these guidelines, containing 8-39
residues covering 75% of the sequence of the influenza virus
hemagglutinin HA1 polypeptide chain, induced antibodies that
reacted with the HA1 protein or intact virus; and 12/12 peptides
from the MuLV polymerase and 18/18 from the rabies glycoprotein
induced antibodies that precipitated the respective proteins.
[0099] Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention. Thus, a high proportion of hybridomas obtained by
fusion of spleen cells from donors immunized with an antigen
epitope-bearing peptide generally secrete antibody reactive with
the native protein. Sutcliffe et al., supra, at 663. The antibodies
raised by antigenic epitope-bearing peptides or polypeptides are
useful to detect the mimicked protein, and antibodies to different
peptides may be used for tracking the fate of various regions of a
protein precursor which undergoes post-translational processing.
The peptides and anti-peptide antibodies may be used in a variety
of qualitative or quantitative assays for the mimicked protein, for
instance in competition assays since it has been shown that even
short peptides (e.g., about 9 amino acids) can bind and displace
the larger peptides in immunoprecipitation assays. See, for
instance, Wilson et al., Cell 37:767-778 (1984) at 777. The
anti-peptide antibodies of the invention also are useful for
purification of the mimicked protein, for instance, by adsorption
chromatography using methods well known in the art.
[0100] Antigenic epitope-bearing peptides and polypeptides of the
invention designed according to the above guidelines preferably
contain a sequence of at least seven, more preferably at least nine
and most preferably between about 15 to about 30 amino acids
contained within the amino acid sequence of a polypeptide of the
invention. However, peptides or polypeptides comprising a larger
portion of an amino acid sequence of a polypeptide of the
invention, containing about 30 to about 50 amino acids, or any
length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are considered epitope-bearing
peptides or polypeptides of the invention and also are useful for
inducing antibodies that react with the mimicked protein.
Preferably, the amino acid sequence of the epitope-bearing peptide
is selected to provide substantial solubility in aqueous solvents
(i.e., the sequence includes relatively hydrophilic residues and
highly hydrophobic sequences are preferably avoided); and sequences
containing proline residues are particularly preferred.
[0101] Non-limiting examples of antigenic polypeptides or peptides
that can be used to generate PAPAI-specific antibodies include: a
polypeptide comprising amino acid residues from about 6 to about 16
in SEQ ID NO:2; a polypeptide comprising amino acid residues from
about 31 to about 36 in SEQ ID NO:2; a polypeptide comprising amino
acid residues from about 46 to about 76 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about 111 to about
121 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 146 to about 161 in SEQ ID NO:2; a polypeptide
comprising amino acid residues from about 206 to about 211 in SEQ
ID NO:2; a polypeptide comprising amino acid residues from about
236 to about 246 in SEQ ID NO:2; a polypeptide comprising amino
acid residues from about 306 to about 316 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about 361 to about
366 in SEQ ID NO:2; a polypeptide comprising amino acid residues
from about 2 to about 12 in SEQ ID NO:13; a polypeptide comprising
amino acid residues from about 27 to about 32 in SEQ ID NO:13; a
polypeptide comprising amino acid residues from about 42 to about
72 in SEQ ID NO:13; a polypeptide comprising amino acid residues
from about 107 to about 117 in SEQ ID NO:13; a polypeptide
comprising amino acid residues from about 142 to about 157 in SEQ
ID NO:13; a polypeptide comprising amino acid residues from about
202 to about 207 in SEQ ID NO:13; a polypeptide comprising amino
acid residues from about 232 to about 242 in SEQ ID NO:13; a
polypeptide comprising amino acid residues from about 302 to about
312 in SEQ ID NO:13; and a polypeptide comprising amino acid
residues from about 357 to about 362 in SEQ ID NO:13. As indicated
above, the inventors have determined that the above polypeptide
fragments are antigenic regions of the PAPAI gene.
[0102] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means for making
peptides or polypeptides including recombinant means using nucleic
acid molecules of the invention. For instance, a short
epitope-bearing amino acid sequence may be fused to a larger
polypeptide which acts as a carrier during recombinant production
and purification, as well as during immunization to produce
anti-peptide antibodies. Epitope-bearing peptides also may be
synthesized using known methods of chemical synthesis. For
instance, Houghten has described a simple method for synthesis of
large numbers of peptides, such as 10-20 mg of 248 different 13
residue peptides representing single amino acid variants of a
segment of the HA1 polypeptide which were prepared and
characterized (by ELISA-type binding studies) in less than four
weeks. Houghten, R. A. (1985) General method for the rapid
solid-phase synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986). In this
procedure the individual resins for the solid-phase synthesis of
various peptides are contained in separate solvent-permeable
packets, enabling the optimal use of the many identical repetitive
steps involved in solid-phase methods. A completely manual
procedure allows 500-1000 or more syntheses to be conducted
simultaneously. Houghten et al., supra, at 5134.
[0103] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).
Generally, animals may be immunized with free peptide; however,
anti-peptide antibody titer may be boosted by coupling of the
peptide to a macromolecular carrier, such as keyhole limpet
hemacyanin (KLH) or tetanus toxoid. For instance, peptides
containing cysteine may be coupled to carrier using a linker such
as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other
peptides may be coupled to carrier using a more general linking
agent such as glutaraldehyde. Animals such as rabbits, rats and
mice are immunized with either free or carrier-coupled peptides,
for instance, by intraperitoneal and/or intradermal injection of
emulsions containing about 100 .mu.g peptide or carrier protein and
Freund's adjuvant. Several booster injections may be needed, for
instance, at intervals of about two weeks, to provide a useful
titer of anti-peptide antibody which can be detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The
titer of anti-peptide antibodies in serum from an immunized animal
may be increased by selection of anti-peptide antibodies, for
instance, by adsorption to the peptide on a solid support and
elution of the selected antibodies according to methods well known
in the art.
[0104] Immunogenic epitope-bearing peptides of the invention, i.e.,
those parts of a protein that elicit an antibody response when the
whole protein is the immunogen, are identified according to methods
known in the art. For instance, Geysen et al., supra, discloses a
procedure for rapid concurrent synthesis on solid supports of
hundreds of peptides of sufficient purity to react in an
enzyme-linked immunosorbent assay. Interaction of synthesized
peptides with antibodies is then easily detected without removing
them from the support. In this manner a peptide bearing an
immunogenic epitope of a desired protein may be identified
routinely by one of ordinary skill in the art. For instance, the
immunologically important epitope in the coat protein of
foot-and-mouth disease virus was located by Geysen et al. with a
resolution of seven amino acids by synthesis of an overlapping set
of all 208 possible hexapeptides covering the entire 213 amino acid
sequence of the protein. Then, a complete replacement set of
peptides in which all 20 amino acids were substituted in turn at
every position within the epitope were synthesized, and the
particular amino acids conferring specificity for the reaction with
antibody were determined. Thus, peptide analogs of the
epitope-bearing peptides of the invention can be made routinely by
this method. U.S. Pat. No. 4,708,781 to Geysen (1987) further
describes this method of identifying a peptide bearing an
immunogenic epitope of a desired protein.
[0105] Further still, U.S. Pat. No. 5,194,392 to Geysen (1990)
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a "mimotope") which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,433,092 to
Geysen (1989) describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand binding site of a
particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971
to Houghten, R. A. et al. (1996) on Peralkylated Oligopeptide
Mixtures discloses linear C.sub.1-C.sub.7-alkyl peralkylated
oligopeptides and sets and libraries of such peptides, as well as
methods for using such oligopeptide sets and libraries for
determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
[0106] The entire disclosure of each document cited in this section
on "Polypeptides and Peptides" is hereby incorporated herein by
reference.
[0107] As one of skill in the art will appreciate, PAPAI
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. This has been shown, e.g., for
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EPA 394,827;
Traunecker et al., Nature 331:84-86 (1988)). Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG part can
also be more efficient in binding and neutralizing other molecules
than the monomeric PAPAI protein or protein fragment alone
(Fountoulakis et al., J. Biochem 270:3958-3964 (1995)).
Diagnostic and Prognostic Applications of PAPAI
[0108] The present inventors believe that PAPAI is involved in
inhibition of the plasminogen activator system. For a number of
pathologic disorders, including tumor invasion and metastasis,
inflammation, and complications of pregnancy, it is believed that
significantly altered levels of PAPAI gene expression can be
detected in body tissue or fluids taken from an individual having
such a disorder. The level of PAPAI gene expression is measured
relative to a "standard" PAPAI gene expression level, i.e., the
PAPAI expression level in tissue or fluids from an individual not
having the pathologic disorder. Thus, the invention provides a
diagnostic method useful during diagnosis of a pathologic disorder,
which involves assaying the expression level of the gene encoding
the PAPAI protein in tissue or body fluid from an individual and
comparing the gene expression level with a standard PAPAI gene
expression level, whereby an increase or decrease in the gene
expression level over the standard is indicative of a pathologic
disorder.
[0109] For example, substantial alterations in PAPAI expression or
activity can serve as markers of tumor invasiveness and metastasis.
This is because PAPAI regulates the fibrinolytic system.
Angiogenesis, the growth of new vascular tissue, is regulated by a
balance between coagulation and fibrinolysis. Angiogenesis is
essential for the expansion of a primary tumor and is also required
for the growth of established metastases at distant sites
(Holmgren, L. et al., Nature Medicine 1: 149-153 (1995)). The level
of PAI-1 in tumors indicates the level of vascularization, and
highly vascularized tumors have higher chances of invasion and
metastasis (Fazioli, F. et al., Trends Pharm. Sci. 15:25-29
(1995)). Overexpression of PAI-2 in malignant melanoma cells
inhibits metastasis in vivo (Mueller, B. M. et al., Pro. Natl.
Acad. Sci. USA 92:205-209 (1995)). The present inventors have shown
that PAPAI is expressed in myoepithelial cells surrounding normal
mammary glands and in benign lesions, but not in infiltrating
breast carcinomas. Thus, the invention provides a method for
predicting whether a tumor is likely to remain stable or will
invade tissue and ultimately metastasize. As a result, decisions
about whether to pursue relatively invasive clinical interventions,
such as surgery, rather than relatively non-invasive interventions,
such as chemotherapy and radiation therapy, can be rationally made.
The terms "tumor invasiveness" and metastasis are well understood
by those of ordinary skill in the art. For example, see Holmgren et
al., Fazioli et al., and Mueller et al., supra.
[0110] Substantial alterations in PAPAI expression or activity can
be used to predict whether hemorrhage is likely to occur in
patients who suffer from hepatic illnesses. Alcoholic cirrhosis,
primary biliary cirrhosis, and liver cancer are all diseases which
are accompanied by hemorrhage due to fibrinolytic bleeding. That
is, the bleeding which occurs is not due to injury, but rather is
due to a dysregulated fibrinolytic system. The overall level of
plasminogen activator activity represents a balance between the
relative levels of activator and inhibitor. Changes in PAPAI
activity, or a difference in the ratio of plasminogen activator to
PAPAI can serve as indicators of imminent hemorrhage in patients
who suffer from alcoholic cirrhosis, primary biliary cirrhosis, and
liver cancer. Thus, the invention further provides a method for
predicting whether hemorrhage will occur in such patients. The
terms "hemorrhage," "alcoholic cirrhosis," "primary biliary
cirrhosis," "liver cancer, and "fibrinolytic bleeding" are well
understood by those of ordinary skill in the art. For example, see
Leiper, K. et al., J. Clin. Pathol. 47:214-217 (1994).
[0111] Substantial alterations in PAPAI expression or activity can
be used to predict whether a patient is likely to develop
preeclampsia. Preeclampsia is a clinical syndrome that affects
women in the third trimester of pregnancy. The syndrome is
characterized by hypertension and proteinuria. The etiology of this
obstetric complication is unknown. However, it is associated with
fibrin deposition in the subendothelium of the renal glomerulus and
in the decidua segments of spiral arteries. In fatal cases of
eclampsia, widespread fibrin deposition has been a prominent
histologic finding. Changes in PAPAI activity, or a difference in
the ratio of plasminogen activator to PAPAI can serve as indicators
of an imminent advance from the pre-eclamptic to the eclamptic
state in patients who are at risk for eclampsia. Thus, the
invention further provides a method for predicting whether a
pre-eclamptic patient is at risk for developing eclampsia. The term
"preeclampsia" is well understood by those of ordinary skill in the
art. For example, see Koh, C. L. et al., Gynecol. Obstet. Invest.
35:214-221 (1993).
[0112] Alterations in PAPAI expression can be assayed at the level
of messenger RNA transcription or protein expression. Suitable
assay techniques are disclosed below. Alternatively, PAPAI
inhibitory activity can be assayed using a spectrophotometric
plasminogen activator inhibitor assay. Such an assay is well-known
to those of ordinary skill in the art. For example, see Erikkson,
E. et al., Thrombosis Research 50:91-101 (1988).
[0113] By individual is intended mammalian individuals, preferably
humans. By "measuring the expression level of the gene encoding the
PAPAI protein" is intended qualitatively or quantitatively
measuring or estimating the level of the PAPAI or the level of the
mRNA encoding the PAPAI protein in a first biological sample either
directly (e.g., by determining or estimating absolute protein level
or mRNA level) or relatively (e.g., by comparing to the PAPAI
protein level or mRNA level in a second biological sample).
Preferably, the PAPAI protein level or mRNA level in the first
biological sample is measured or estimated and compared to a
standard PAPAI protein level or mRNA level, the standard being
taken from a second biological sample obtained from an individual
not having the disorder or being determined by averaging levels
from a population of individuals not having the disorder. As will
be appreciated in the art, once a standard PAPAI protein level or
mRNA level is known, it can be used repeatedly as a standard for
comparison.
[0114] By "biological sample" is intended any biological sample
obtained from an individual, a cell line, a tissue culture, or
other source which contains PAPAI protein or mRNA, secretes mature
PAPAI protein, or expresses the PAPAI receptor. Biological samples
include normal tissue or cells and tumor cells (whether malignant
or benign). Biological samples include body fluids, including whole
blood, serum, plasma, urine, saliva, tears, pulmonary secretions,
gastrointestinal secretions, fecal material, lymph fluid, synovial
fluid, and cerebrospinal fluid. Methods for obtaining tissue
biopsies and body fluids from mammals are well known in the art.
Where the biological sample is to include mRNA, a tissue biopsy is
the preferred source.
[0115] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described in
Chomczynski and Sacchi, Anal. Biochem. 162.156-159 (1987). Levels
of mRNA encoding the PAPAI protein are then assayed using any
appropriate method. These include Northern blot analysis, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription (RT) in combination with the polymerase chain
reaction (RT-PCR), and reverse transcription in combination with
the ligase chain reaction (RT-LCR).
[0116] Northern blot analysis can be performed as described in
Harada et al., Cell 63:303-312 (1990). Briefly, total RNA is
prepared from a biological sample as described above. For the
Northern blot, the RNA is denatured in an appropriate buffer (such
as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subjected
to agarose gel electrophoresis, and transferred onto a
nitrocellulose filter. After the RNAs have been linked to the
filter by a UV linker, the filter is prehybridized in a solution
containing formamide, SSC, Denhardt's solution, denatured salmon
sperm, SDS, and sodium phosphate buffer. PAPAI protein cDNA labeled
according to any appropriate method (such as the
.sup.32P-multiprimed DNA labeling system (Amersham)) is used as
probe. After hybridization overnight, the filter is washed and
exposed to x-ray film. cDNA for use as probe according to the
present invention is described in the sections above and will
preferably at least 15 bp in length.
[0117] S1 mapping can be performed as described in Fujita et al.,
Cell 49:357-367 (1987). To prepare probe DNA for use in S1 mapping,
the sense strand of above-described cDNA is used as a template to
synthesize labeled antisense DNA. The antisense DNA can then be
digested using an appropriate restriction endonuclease to generate
further DNA probes of a desired length. Such antisense probes are
useful for visualizing protected bands corresponding to the target
mRNA (i.e., mRNA encoding the PAPAI protein). Northern blot
analysis can be performed as described above.
[0118] Preferably, levels of mRNA encoding the PAPAI protein are
assayed using the RT-PCR method described in Makino et al.,
Technique 2.295-301 (1990). By this method, the radioactivities of
the "amplicons" in the polyacrylamide gel bands are linearly
related to the initial concentration of the target mRNA. Briefly,
this method involves adding total RNA isolated from a biological
sample in a reaction mixture containing a RT primer and appropriate
buffer. After incubating for primer annealing, the mixture can be
supplemented with a RT buffer, dNTPs, DTT, RNase inhibitor and
reverse transcriptase. After incubation to achieve reverse
transcription of the RNA, the RT products are then subject to PCR
using labeled primers. Alternatively, rather than labeling the
primers, a labeled dNTP can be included in the PCR reaction
mixture. PCR amplification can be performed in a DNA thermal cycler
according to conventional techniques. After a suitable number of
rounds to achieve amplification, the PCR reaction mixture is
electrophoresed on a polyacrylamide gel. After drying the gel, the
radioactivity of the appropriate bands (corresponding to the mRNA
encoding the PAPAI protein) is quantified using an imaging
analyzer. RT and PCR reaction ingredients and conditions, reagent
and gel concentrations, and labeling methods are well known in the
art. Variations on the RT-PCR method will be apparent to the
skilled artisan.
[0119] Any set of oligonucleotide primers which will amplify
reverse transcribed target mRNA can be used and can be designed as
described in the sections above.
[0120] Assaying PAPAI protein levels in a biological sample can
occur using any art-known method. Preferred for assaying PAPAI
protein levels in a biological sample are antibody-based
techniques. For example, PAPAI protein expression in tissues can be
studied with classical immunohistological methods. In these, the
specific recognition is provided by the primary antibody
(polyclonal or monoclonal) but the secondary detection system can
utilize fluorescent, enzyme, or other conjugated secondary
antibodies. As a result, an immunohistological staining of tissue
section for pathological examination is obtained. Tissues can also
be extracted, e.g., with urea and neutral detergent, for the
liberation of PAPAI protein for Western-blot or dot/slot assay
(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985));
Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). In this
technique, which is based on the use of cationic solid phases,
quantitation of PAPAI protein can be accomplished using isolated
PAPAI protein as a standard. This technique can also be applied to
body fluids. With these samples, a molar concentration of PAPAI
protein will aid to set standard values of PAPAI protein content
for different body fluids, like serurn, plasma, urine, synovial
fluid, spinal fluid, etc. The normal appearance of PAPAI protein
amounts can then be set using values from healthy individuals,
which can be compared to those obtained from a test subject.
[0121] Other antibody-based methods useful for detecting PAPAI
protein levels include immunoassays, such as the enzyme linked
immunoadsorbent assay (ELISA) and the radioimmunoassay (RIA). For
example, PAPAI protein-specific monoclonal antibodies can be used
both as an immunoadsorbent and as an enzyme-labeled probe to detect
and quantify the PAPAI protein. The amount of PAPAI protein present
in the sample can be calculated by reference to the amount present
in a standard preparation using a linear regression computer
algorithm. Such an ELISA for detecting a tumor antigen is described
in lacobelli et al., Breast Cancer Research and Treatment 11:19-30
(1988). In another ELISA assay, two distinct specific monoclonal
antibodies can be used to detect PAPAI protein in a body fluid. In
this assay, one of the antibodies is used as the immunoadsorbent
and the other as the enzyme-labeled probe.
[0122] The above techniques may be conducted essentially as a
"one-step" or "two-step" assay. The "one-step" assay involves
contacting PAPAI protein with immobilized antibody and, without
washing, contacting the mixture with the labeled antibody. The
"two-step" assay involves washing before contacting the mixture
with the labeled antibody. Other conventional methods may also be
employed as suitable. It is usually desirable to immobilize one
component of the assay system on a support, thereby allowing other
components of the system to be brought into contact with the
component and readily removed from the sample.
[0123] Suitable enzyme labels include, for example, those from the
oxidase group, which catalyze the production of hydrogen peroxide
by reacting with substrate. Glucose oxidase is particularly
preferred as it has good stability and its substrate (glucose) is
readily available. Activity of an oxidase label may be assayed by
measuring the concentration of hydrogen peroxide formed by the
enzyme-labeled antibody/substrate reaction. Besides enzymes, other
suitable labels include radioisotopes, such as iodine (.sup.125I,
.sup.121I), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.112In), and technetium (.sup.99mTc), and
fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0124] In addition to assaying PAPAI protein levels in a biological
sample obtained from an individual, PAPAI protein can also be
detected in vivo by imaging. Antibody labels or markers for in vivo
imaging of PAPAI protein-include those detectable by X-radiography,
NMR or ESR. For X-radiography, suitable labels include
radioisotopes such as barium or cesium, which emit detectable
radiation but are not overtly harmful to the subject. Suitable
markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma.
[0125] A PAPAI protein-specific antibody or antibody portion which
has been labeled with an appropriate detectable imaging moiety,
such as a radioisotope (for example, .sup.131I, .sup.112In,
.sup.99mTc), a radio-opaque substance, or a material detectable by
nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the mammal
to be examined. It will be understood in the art that the size of
the subject and the imaging system used will determine the quantity
of imaging moieties needed to produce diagnostic images. In the
case of a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of .sup.99mTc. The labeled antibody or antibody portion
will then preferentially accumulate at the location of cells which
contain PAPAI protein. In vivo tumor imaging is described in S. W.
Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies
and Their Portions" (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, eds., S. W. Burchiel and B. A. Rhodes, Masson
Publishing Inc. (1982)).
[0126] PAPAI protein-specific antibodies for use in the present
invention can be raised against the intact PAPAI protein or an
antigenic polypeptide portion thereof, which may presented together
with a carrier protein, such as an albumin, to an animal system
(such as rabbit or mouse) or, if it is long enough (at least about
25 amino acids), without a carrier.
[0127] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody portions (such as, for example, Fab and F(ab').sub.2
portions) which are capable of specifically binding to PAPAI
protein. Fab and F(ab').sub.2 portions lack the Fc portion of
intact antibody, clear more rapidly from the circulation, and may
have less non-specific tissue binding of an intact antibody (Wahl
et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these portions are
preferred.
[0128] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
PAPAI protein or an antigenic portion thereof can be administered
to an animal in order to induce the production of sera containing
polyclonal antibodies. In a preferred method, a preparation of
PAPAI protein is prepared and purified as described above to render
it substantially free of natural contaminants. Such a preparation
is then introduced into an animal in order to produce polyclonal
antisera of greater specific activity.
[0129] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or PAPAI protein binding
portions thereof). Such monoclonal antibodies can be prepared using
hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler
et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J.
Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies
and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In
general, such procedures involve immunizing an animal (preferably a
mouse) with a PAPAI protein antigen or, more preferably, with a
PAPAI protein-expressing cell. Suitable cells can be recognized by
their capacity to bind PAPAI protein antibody. Such cells may be
cultured in any suitable tissue culture medium; however, it is
preferable to culture cells in Earle's modified Eagle's medium
supplemented with 10% fetal bovine serum (inactivated at about
56.degree. C.), and supplemented with about 10 .mu.g/l of
nonessential amino acids, about 1,000 U/ml of penicillin, and about
100 .mu.g/ml of streptomycin. The splenocytes of such mice are
extracted and fused with a suitable myeloma cell line. Any suitable
myeloma cell line may be employed in accordance with the present
invention; however, it is preferable to employ the parent myeloma
cell line (SP.sub.2O), available from the American Type Culture
Collection, Rockville, Md. After fusion, the resulting hybridoma
cells are selectively maintained in HAT medium, and then cloned by
limiting dilution as described by Wands et al. (Gastroenterology
80:225-232 (1981)). The hybridoma cells obtained through such a
selection are then assayed to identify clones which secrete
antibodies capable of binding the PAPAI antigen.
[0130] Alternatively, additional antibodies capable of binding to
the PAPAI protein antigen may be produced in a two-step procedure
through the use of anti-idiotypic antibodies. Such a method makes
use of the fact that antibodies are themselves antigens, and
therefore it is possible to obtain an antibody which binds to a
second antibody. In accordance with this method, PAPAI protein
specific antibodies are used to immunize an animal, preferably a
mouse. The splenocytes of such an animal are then used to produce
hybridoma cells, and the hybridoma cells are screened to identify
clones which produce an antibody whose ability to bind to the PAPAI
protein-specific antibody can be blocked by the PAPAI protein
antigen. Such antibodies comprise anti-idiotypic antibodies to the
PAPAI protein-specific antibody and can be used to immunize an
animal to induce formation of further PAPAI protein-specific
antibodies.
[0131] It will be appreciated that Fab and F(ab').sub.2 and other
portions of the antibodies of the present invention may be used
according to the methods disclosed herein. Such portions are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab portions) or pepsin (to produce F(ab').sub.2
portions). Alternatively, PAPAI protein-binding portions can be
produced through the application of recombinant DNA technology or
through synthetic chemistry.
[0132] Where in vivo imaging is used to detect enhanced levels of
PAPAI protein for diagnosis in humans, it may be preferable to use
"humanized" chimeric monoclonal antibodies. Such antibodies can be
produced using genetic constructs derived from hybridoma cells
producing the monoclonal antibodies described above. Methods for
producing chimeric antibodies are known in the art. See, for
review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4.214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et
al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
8601533; Robinson et al., WO 8702671; Boulianne et al., Nature
312:643 (1984); Neuberger et al., Nature 314:268 (1985).
[0133] Further suitable labels for the PAPAI protein-specific
antibodies of the present invention are provided below. Examples of
suitable enzyme labels include malate dehydrogenase, staphylococcal
nuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase,
alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,
peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase, and acetylcholine
esterase.
[0134] Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47SC,
.sup.109Pd, etc. .sup.111In is a preferred isotope where in vivo
imaging is used since its avoids the problem of dehalogenation of
the .sup.125I, or .sup.131I-labeled monoclonal antibody by the
liver. In addition, this radio nucleotide has a more favorable
gamma emission energy for imaging (Perkins et al., Eur. J. Nucl.
Med. 10:296-301 (1985); Carasquillo et al., J Nucl. Med. 28:281-287
(1987)). For example, .sup.111In coupled to monoclonal antibodies
with 1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in
non-tumorous tissues, particularly the liver, and therefore
enhances specificity of tumor localization (Esteban et al., J.
Nucl. Med. 28:861-870 (1987)).
[0135] Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, and .sup.56Fe.
[0136] Examples of suitable fluorescent labels include an
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, an o-phthaldehyde label, and a fluorescamine
label.
[0137] Examples of suitable toxin labels include diphtheria toxin,
ricin, and cholera toxin.
[0138] Examples of chemiluminescent labels include a luminal label,
an isoluminal label, an aromatic acridinium ester label, an
imidazole label, an acridinium salt label, an oxalate ester label,
a luciferin label, a luciferase label, and an aequorin label.
[0139] Examples of nuclear magnetic resonance contrasting agents
include heavy metal nuclei such as Gd, Mn, and Fe.
[0140] Typical techniques for binding the above-described labels to
antibodies are provided by Kennedy et al. (Clin. Chim. Acta 70:1-31
(1976)), and Schurs et al. (Clin. Chim. Acta 81:1-40(1977)).
Coupling techniques mentioned in the latter are the glutaraldehyde
method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which
methods are incorporated by reference herein.
Chromosome Assays
[0141] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0142] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a PAPAI protein
gene. This can be accomplished using a variety of well known
techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome
mapping using well known techniques for this purpose. Typically, in
accordance with routine procedures for chromosome mapping, some
trial and error may be necessary to identify a genomic probe that
gives a good in situ hybridization signal.
[0143] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Only those
hybrids containing the human gene corresponding to the primer will
yield an amplified portion.
[0144] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of portions from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0145] Fluorescence in situ hybridization ("FISH") of a cDNA clone
to a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
probes from the cDNA as short as 50 or 60 bp. For a review of this
technique, see Verma et al., Human Chromosomes: A Manual OfBasic
Techniques, Pergamon Press, New York (1988).
[0146] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance In Man, available
on-line through Johns Hopkins University, Welch Medical Library.
The relationship between genes and diseases that have been mapped
to the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0147] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0148] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. This assumes 1 megabase mapping
resolution and one gene per 20 kb.
Therapeutic Uses of PAPAI
[0149] As discussed above, the PAPAI-protein is believed to be
involved in inhibition of the plasminogen activator system and
plays a role in a wide variety of physiologic and pathologic
processes. Accordingly, the PAPAI protein has application to any
physiologic or pathologic disease condition in which abnormal
activity of the plasminogen activator system is implicated and has
pathological or physiological consequences. A large number of
disease conditions are associated with modifications of the
plasminogen activator system (Kruithof et al., Thromb. Haemost.
59:7 (1988)). Examples of such disease conditions include, but are
not limited to: complications of pregnancy, such as preeclampsia
and intrauterine growth retardation (Halligan et al., Br. Obstet.
Gyneco., 101:488 (1994); Gilabert et al., Gynecol. Obstet. Invest.
38:157(1994)); cancer (Dano et al., Fibrinolysis 8:189 (1994);
Fazoli, F. et al., Trends Pharmacol. Sci. 15:25 (1994); Shinkfield
et al., Fibrinolysis 6:59 (1992)); and wound healing (Schafer et
al., Amer. J Pathol. 144:1269 (1994)). Because of the role of the
plasminogen activator system in these disease states, inhibition of
the PA system by PAPAI should provide therapeutic benefits to an
individual suffering from one (or more) of these physiologic or
pathologic diseases.
[0150] Given the fibrinolytic activities modulated by PAPAI, it is
readily apparent that a substantially altered level of expression
of PAPAI in an individual, compared to the standard or "normal"
level, produces pathological conditions such as those described
above in relation to diagnosis. It will also be appreciated by one
of ordinary skill that, since the PAPAI protein of the invention is
translated with a leader peptide suitable for secretion of the
mature protein from the cells which express PAPAI, when PAPAI
protein (particularly the mature form) is added from an exogenous
source to cells, tissues or the body of an individual, the protein
will exert its modulating activities on any of its target cells of
that individual. Therefore, it will be appreciated that conditions
caused by a decrease in the standard or normal level of PAPAI
activity in an individual, can be treated by administration of
PAPAI protein. Thus, the invention also provides a method of
treatment of an individual in need of an increased level of
activity comprising administering to such an individual a
pharmaceutical composition comprising an amount of an isolated
PAPAI polypeptide of the invention, particularly a mature form of
the PAPAI protein of the invention, effective to increase the PAPAI
activity level in such an individual.
[0151] For example, since plasminogen activator inhibitors inhibit
tumor cell invasion and metastasis, the invention provides a method
for treating or preventing tumor invasion and metastasis in cancers
including, but not limited to, leukemia, lung cancer, breast
cancer, endometrial and ovarian cancer, melanoma, and
gastrointestinal cancers, including pancreatic cancer and
colorectal cancer. The present inventors have shown, in an animal
model, that the presence of PAPAI alters the invasive potential of
breast cancer cells.
[0152] In addition, since plasminogen activator inhibitors inhibit
fibrinolysis, the invention provides a method for treating or
preventing coagulation disorders including, but not limited to,
arterial thrombi, venous thrombi, disseminated intravascular
coagulation, and excessive bleeding caused by the administration of
a pharmaceutical plasminogen activator (such as urokinase or tissue
plasminogen activator).
[0153] Further, since protease inhibitors are effective antiviral
agents, the invention provides a method for treating or preventing
infections caused by viruses including, but not limited to, Human
Immunodeficiency Virus 1 (HIV-1), HIV-2, hepatitis A, hepatitis B,
hepatitis C, hepatitis E, hepatitis F, and hepatitis G.
[0154] One of ordinary skill will appreciate that effective amounts
of the PAPAI polypeptides for treating an individual in need of an
increased level of PAPAI activity (including amounts of PAPAI
polypeptides effective for the conditions discussed above, with or
without other or plasminogen activator inhibitors or other agents)
can be determined empirically for each condition where
administration of PAPAI is indicated.
[0155] The polypeptide having PAPAI activity may be administered in
pharmaceutical compositions in combination with one or more
pharmaceutically acceptable excipients. It will be understood that,
when administered to a human patient, the total daily usage of the
pharmaceutical compositions of the present invention will be
decided by the attending physician within the scope of sound
medical judgment. The specific therapeutically effective dose level
for any particular patient will depend upon a variety of factors
including the type and degree of the response to be achieved; the
specific composition an other agent, if any, employed; the age,
body weight, general health, sex and diet of the patient; the time
of administration, route of administration, and rate of excretion
of the composition; the duration of the treatment; drugs (such as a
chemotherapeutic agent) used in combination or coincidental with
the specific composition; and like factors well known in the
medical arts.
[0156] The PAPAI composition to be used in the therapy will be
formulated and dosed in a fashion consistent with good medical
practice, taking into account the clinical condition of the
individual patient (especially the side effects of treatment with
PAPAI alone), the site of delivery of the PAPAI composition, the
method of administration, the scheduling of administration, and
other factors known to practitioners. The "effective amount" of
PAPAI for purposes herein (including a PAPAI effective amount) is
thus determined by such considerations.
[0157] As a general proposition, the total pharmaceutically
effective amount of the PAPAI administered parenterally per dose
will be in the range of about 1 .mu.g/kg/day to 10 mg/kg/day of
patient body weight, although, as noted above, this will be subject
to therapeutic discretion. More preferably, this dose is at least
0.01 mg/kg/day, and most preferably for humans between about 0.01
and 1 mg/kg/day. If given continuously, the PAPAI is typically
administered at a dose rate of about 1 .mu.g/kg/hour to about 50
.mu.g/kg/hour, either by 1-4 injections per day or by continuous
subcutaneous infusions, for example, using a mini-pump. An
intravenous bag solution or bottle solution may also be
employed.
[0158] A course of PAPAI treatment to affect the fibrinolytic
system appears to be optimal if continued longer than a certain
minimum number of days, 7 days in the case of the mice. The length
of treatment needed to observe changes and the interval following
treatment for responses to occur appears to vary depending on the
desired effect.
[0159] The PAPAI is also suitably administered by sustained-release
systems. Suitable examples of sustained-release compositions
include semi-permeable polymer matrices in the form of shaped
articles, e.g., films, or mirocapsules. Sustained-release matrices
include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U.
Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl
methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15:167-277
(1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (R. Langer et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
PAPAI compositions also include liposomally entrapped PAPAI.
Liposomes containing PAPAI are prepared by methods known per se: DE
3,218,121; Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688-3692
(1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030-4034
(1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;
Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and
4,544,545; and EP 102,324. Ordinarily, the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid
content is greater than about 30 mol. percent cholesterol, the
selected proportion being adjusted for the optimal PAPAI
therapy.
[0160] For parenteral administration, in one embodiment, the PAPAI
is formulated generally by mixing it at the desired degree of
purity, in a unit dosage injectable form (solution, suspension, or
emulsion), with a pharmaceutically acceptable carrier, i.e., one
that is non-toxic to recipients at the dosages and concentrations
employed and is compatible with other ingredients of the
formulation. For example, the formulation preferably does not
include oxidizing agents and other compounds that are known to be
deleterious to polypeptides.
[0161] Generally, the formulations are prepared by contacting the
PAPAI uniformly and intimately with liquid carriers or finely
divided solid carriers or both. Then, if necessary, the product is
shaped into the desired formulation. Preferably the carrier is a
parenteral carrier, more preferably a solution that is isotonic
with the blood of the recipient. Examples of such carrier vehicles
include water, saline, Ringer's solution, and dextrose solution.
Non-aqueous vehicles such as fixed oils and ethyl oleate are also
useful herein, as well as liposomes.
[0162] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0163] PAPAI is typically formulated in such vehicles at a
concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10
mg/ml, at a pH of about 3 to 8. It will be understood that the use
of certain of the foregoing excipients, carriers, or stabilizers
will result in the formation of PAPAI salts.
[0164] PAPAI to be used for therapeutic administration must be
sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes).
Therapeutic PAPAI compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0165] PAPAI ordinarily will be stored in unit or multi-dose
containers, for example, sealed ampules or vials, as an aqueous
solution or as a lyophilized formulation for reconstitution. As an
example of a lyophilized formulation, 10-ml vials are filled with 5
ml of sterile-filtered 1% (w/v) aqueous PAPAI solution, and the
resulting mixture is lyophilized. The infusion solution is prepared
by reconstituting the lyophilized PAPAI using bacteriostatic
Water-for-Injection.
[0166] Dosaging may also be arranged in a patient specific manner
to provide a predetermined concentration of an PAPAI activity in
the blood, as determined by an RIA technique, for instance. Thus
patient dosaging may be adjusted to achieve regular on-going trough
blood levels, as measured by RIA, on the order of from 50 to 1000
ng/ml, preferably 150 to 500 ng/ml.
[0167] Pharmaceutical compositions of the invention may be
administered orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, topically (as by powders,
ointments, drops or transdermal patch), bucally, or as an oral or
nasal spray. By "pharmaceutically acceptable carrier" is meant a
non-toxic solid, semisolid or liquid filler, diluent, encapsulating
material or formulation auxiliary of any type. The term
"parenteral" as used herein refers to modes of administration which
include intravenous, intramuscular, intraperitoneal, intrasternal,
subcutaneous and intraarticular injection and infusion.
[0168] The PAPAI polypeptide may also be employed in accordance
with the present invention by expression of such polypeptides in
vivo or ex vivo, which is often referred to as "gene therapy". Gene
therapy with the PAPAI polypeptide is accomplished by introduction
of a recombinant vector containing a polynucleotide encoding the
PAPAI polypeptide into cells, either in vivo or ex vivo. Methods
for preparing recombinant vectors are well known in the art and
described, for example, in Molecular Cloning, A Laboratory Manual,
2nd. edition, edited by Sambrook, J., Fritsch, E. F. and Maniatis,
T., (1989), Cold Spring Harbor Laboratory Press
[0169] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo,
with the engineered cells then being provided to a patient to be
treated with the polypeptide. Such methods are well-known in the
art. For example, cells may be engineered by procedures known in
the art by infection with a recombinant viral particle encoding a
polypeptide of the present invention. Cells may also be engineered
by other techniques, including introduction of recombinant plasmid
vectors by means of liposomes, electroporation, microinjection, or
calcium phosphate precipitation.
[0170] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by, for example, procedures known in the art.
A recombinant viral or plasmid vector containing the polynucleotide
encoding the PAPAI polypeptide is introduced into the target cell.
Viral vectors useful in the invention include retroviral vectors,
adenoviral vectors, HSV-based vector systems, vaccinia vectors,
papovaviruses, and adeno-associated virus.
[0171] Delivery of retroviral vectors can be accomplished either by
direct infection of target; or by injection of a viral producing
cell line for replication-deficient retroviruses, which provides a
continuous source of vector particles. Plasmid vectors can be
administered in vivo by liposomes, direct injection, and
receptor-mediated nucleic acid transfer. Preferred delivery methods
include those that target the specific tissue to be treated. For
example, for the treatment of breast cancer, receptor-mediated gene
transfer by conjugation of the plasmid vector to estrogen via
polylysine is one preferred method of delivery (EP 785216).
[0172] The nucleic acid sequence encoding the polypeptide of the
present invention is under the control of a suitable promoter.
Suitable promoters which may be employed include, but are not
limited to, the retroviral LTR; the SV40 promoter; the human
cytomegalovirus (CMV) promoter; adenovirus promoters; the
respiratory syncytial virus promoter; thymidine kinase promoters;
inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters, and cellular promoters including,
but not limited to, the histone, pol 111, and P-actin promoters.
Preferred promoters are those that are active only in the target
cell. For example, for treatment of breast cancer, preferred
promoters include the DF3/MUC1 promoter (Manome, Y. et al., J.
Biol. Che, 271: 10560-10568 (1996), the mouse mammary tumor virus
long terminal repeat (Holt et al., Human Gene Therapy 7:1367-1380
(1996), and the whey acid protein promoter (Doppler, W. et al.,
Mol. Endocrinol. 5:1524-1632 (1991)).
[0173] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
Expression and Purification of PAPAI in E. coli
[0174] The DNA sequence encoding the mature PAPAI protein in the
deposited cDNA clone is amplified using PCR oligonucleotide primers
specific to the amino acid carboxyl terminal sequence of the PAPAI
protein and to vector sequences 3' to the gene. Additional
nucleotides containing restriction sites to facilitate cloning are
added to the 5' and 3' sequences, respectively.
[0175] The 5' oligonucleotide primer has the sequence 5'CGC CCA TGG
GAA GTC AAG CCT CAA G 3' (SEQ ID NO:5) containing the underlined
Nco I restriction site (which encodes a start ATG within the Nco I
site), followed by 16 nucleotides complementary to bp 110-125 of
the antisense strand of the PAPAI protein coding sequence set out
in FIGS. 1A-1B (SEQ ID NO:1).
[0176] The 3' primer has the sequence 5'CGC AAG CTT TCA CTT CCT TTT
ATC TCC CTG 3' (SEQ ID NO:6) containing the underlined Hind III
restriction site, followed by 8 nucleotides complementary to bp
1250-1267 of the sense strand of the PAPAI protein coding sequence
set out in FIGS. 1A-1B (SEQ ID NO:1), and a stop codon.
[0177] The restrictions sites are convenient to restriction enzyme
sites in the bacterial expression vector pQE-60, which is used for
bacterial expression in these examples. (Qiagen, Chatsworth,
Calif., 91311).
[0178] The amplified PAPAI protein DNA and the vector pQE-60 are
both digested with Nco I and Hind III and the digested DNAs are
subsequently ligated together. Insertion of the PAPAI protein DNA
into the pQE-60 restricted vector places the PAPAI protein coding
region downstream of and operably linked to the vector's promoter
and in-frame with an initiating AUG appropriately positioned for
translation of PAPAI protein.
[0179] The ligation mixture is transformed into competent E. coli
cells using standard procedures. Such procedures are described, for
example, in Sambrook et al., Molecular Cloning. A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1989). E. coli strain M15/rep4, containing multiple
copies of the plasmid pREP4, which expresses lac repressor and
confers kanamycin resistance ("Kan.sup.r"), is used in carrying out
the illustrative example described here. This strain, which is only
one of many that are suitable for expressing PAPAI protein, is
available commercially from Qiagen.
[0180] Transformants are identified by their ability to grow on LB
plates in the presence of ampicillin. Plasmid DNA is isolated from
resistant colonies and the identity of the cloned DNA is confirmed
by restriction analysis.
[0181] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml).
[0182] The O/N culture is used to inoculate a large culture, at a
dilution of approximately 1:100 to 1:250. The cells are grown to an
optical density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-B-D-thiogalactopyranoside ("IPTG") are then added to a
final concentration of 1 mM to induce transcription from lac
repressor sensitive promoters, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
are then harvested by centrifugation and disrupted, by standard
methods. Inclusion bodies are purified from the disrupted cells
using routine collection techniques, and protein are solubilized
from the inclusion bodies into 8M urea. The 8M urea solution
containing the solubilized protein is passed over a PD-10 column in
2.times. phosphate buffered saline ("PBS"), thereby removing the
urea, exchanging the buffer and refolding the protein. The protein
is purified by a further step of chromatography to remove
endotoxin. Then, it is sterile filtered. The sterile filtered
protein preparation is stored in 2.times.PBS at a concentration of
95 micrograms per mL.
[0183] Analysis of the preparation by standard methods of
polyacrylamide gel electrophoresis reveals that the preparation
contains about 95% monomer PAPAI protein having the expected
molecular weight of approximately 44.5 kDa.
Example 2
Cloning and Expression in Mammalian Cells
[0184] Most of the vectors used for the transient expression of the
PAPAI protein gene sequence in mammalian cells should carry the
SV40 origin of replication. This allows the replication of the
vector to high copy numbers in cells (e.g., COS cells) which
express the T-antigen required for the initiation of viral DNA
synthesis. Any other suitable mammalian cell line can also be
utilized for this purpose.
[0185] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g. RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). Cellular signals may,
however, also be used (e.g., human actin, promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATTC.TM. 37152), pSV2dhfr (ATTC.TM.
37146) and pBCI2MI (ATTC.TM. 67109). Mammalian host cells that may
be used include human Hela, 283, H9 and Jurkart cells, mouse NIH3T3
and C127 cells, Cos 1, Cos 7 and CVI African green monkey cells,
quail QC.sub.1-3 cells, mouse L cells and Chinese hamster ovary
cells.
[0186] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[0187] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
is a useful marker to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful marker is the enzyme glutamine synthase (GS) (Murphy et al.,
Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology
10: 169-175 (1992)). Using these markers, the mammalian cells are
grown in selective medium and the cells with the highest resistance
are selected. These cell lines contain amplified gene(s) integrated
into a chromosome. Chinese hamster ovary (CHO) cells are often used
for the production of proteins.
[0188] The expression vectors pC 1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular
and Cellular Biology 438:4470 (1985)) plus a fragment of the
CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple
cloning sites, such as the restriction enzyme cleavage sites BamHI,
XbaI and Asp718, facilitate the cloning of the gene of interest.
These vectors contain, in addition to the 3' intron, the
polyadenylation and termination signal of the rat preproinsulin
gene.
Example 2(a)
Expression and Purification of Human PAPAI Protein Using the CHO
Expression System
[0189] The vector pC1 is used for the expression of PAPAI protein.
Plasmid pC1 is a derivative of the plasmid pSV2-dhfr (ATTC.TM.
Accession No. 37146). Both plasmids contain the mouse DHFR gene
under control of the SV40 early promoter. Chinese hamster ovary- or
other cells lacking dihydrofolate activity that are transfected
with these plasmids can be selected by growing the cells in a
selective medium (alpha minus MEM, Life Technologies) supplemented
with the chemotherapeutic agent methotrexate. The amplification of
the DHFR genes in cells resistant to methotrexate (MTX) has been
well documented (see, e.g., Alt, F. W., Kellems, R. M., Bertino, J.
R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370, Hamlin,
J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,
Page, M. J. and Sydenharn, M. A. 1991, Biotechnology Vol. 9:64-68).
Cells grown in increasing concentrations of MTX develop resistance
to the drug by overproducing the target enzyme, DHFR, as a result
of amplification of the DHFR gene. If a second gene is linked to
the DHFR gene it is usually co-amplified and over-expressed. It is
state of the art to develop cell lines carrying more than 1,000
copies of the genes. Subsequently, when the methotrexate is
withdrawn, cell lines contain the amplified gene integrated into
the chromosome(s).
[0190] Plasmid pC1 contains for the expression of the gene of
interest a strong promoter of the long terminal repeat (LTR) of the
Rouse Sarcoma Virus (Cullen, et al., Molecular and Cellular
Biology, March 1985:4384470) plus a fragment isolated from the
enhancer of the immediate early gene of human cytomegalovirus (CMV)
(Boshart et al., Cell 41:521-530, 1985). Downstream of the promoter
are the following single restriction enzyme cleavage sites that
allow the integration of the genes: BamHII, PvuII, and NruI. Behind
these cloning sites the plasmid contains translational stop codons
in all three reading frames followed by the 30 intron and the
polyadenylation site of the rat preproinsulin gene. Other high
efficient promoters can also be used for the expression, e.g., the
human P-actin promoter, the SV40 early or late promoters or the
long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
For the polyadenylation of the mRNA other signals, e.g., from the
human growth hormone or globin genes can be used as well.
[0191] Stable cell lines carrying a gene of interest integrated
into the chromosomes can also be selected upon co-transfection with
a selectable marker such as gpt, G418 or hygromycin. It is
advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
[0192] The plasmid pC1 is digested with the restriction enzyme
BamHI and then dephosphorylated using calf intestinal phosphates by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0193] The DNA sequence encoding PAPAI protein in the deposited
polynucleotide is amplified using PCR oligonucleotide primers
specific to the carboxyl terminal sequence of the PAPAI protein and
to vector sequences 3' to the gene. Additional nucleotides
containing restriction sites to facilitate cloning are added to the
5' and 3' sequences respectively.
[0194] The 5' primer has the sequence 5'CGC GGA TCC GCC ATC ATG GAC
ACA ATC TTC TTG 3' (SEQ ID NO:7) containing the underlined Bam HI
restriction enzyme site, followed by 16 nucleotides complementary
to bp 67-84 of the antisense strand of the PAPAI protein coding
sequence set out in FIGS. 1A-1B (SEQ ID NO:1). For the full length
gene, the 3' primer has the full length sequence CGC GGT ACC TCA
CTT CCT TTT ATC TCC CTG (SEQ ID NO:8), containing the underlined
Asp718 restriction site, followed by 8 nucleotides complementary to
bp 1250-1267 of the sense strand of the PAPAI protein coding
sequence set out in FIGS. 1A-1B (SEQ ID NO:1), and a stop
codon.
[0195] The restrictions sites are convenient to restriction enzyme
sites in the CHO expression vector CHO-1. The amplified PAPAI
protein DNA and the vector CHO-1 both are digested with BamHII and
Asp718 and the digested DNAs subsequently ligated together.
Insertion of the PAPAI protein DNA into the BamHI/Asp718 digested
vector places the PAPAI protein coding region downstream of and
operably linked to the vector's promoter. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037) the transformed culture is plated on ampicillin media plates
which then are incubated to allow growth of ampicillin resistant
colonies. Plasmid DNA is isolated from resistant colonies and
examined by restriction analysis and gel sizing for the presence of
the PAPAI-encoding fragment. Transfection of CHO-DHFR-cells
[0196] Chinese hamster ovary cells lacking an active DHFR enzyme
are used for transfection. 5 .mu.g of the expression plasmid C I
are cotransfected with 0.5 .mu.g of the plasmid pSVneo using the
LIPOFECTIN.TM. method (Felgner et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the gene neo from Tn5
encoding an enzyme that confers resistance to a group of
antibiotics including G418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) and cultivated from 10-14 days. After this period, single
clones are trypsinized and then seeded in 6-well petri dishes using
different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200
nM, 400 nM). Clones growing at the highest concentrations of
methotrexate are then transferred to new 6-well plates containing
even higher concentrations of methotrexate (500 nM, 1 .mu.M, 2
.mu.M, 5 .mu.M). The same procedure is repeated until clones grow
at a concentration of 100 .mu.M.
[0197] The expression of the desired gene product is analyzed by
Western blot analysis and SDS-PAGE.
Example 2(b)
Expression and Purification of Human PAPAI Protein Using the COS
Expression System
[0198] The expression plasmid, pPAPAI HA, is made by cloning a cDNA
encoding PAPAI into the expression vector pcDNAI/Amp (which can be
obtained from Invitrogen, Inc.).
[0199] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron, and a polyadenylation
signal arranged so that a cDNA conveniently can be placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker.
[0200] A DNA fragment encoding the entire PAPAI precursor and an HA
tag fised in frame to its 3' end is cloned into the polylinker
region of the vector so that recombinant protein expression is
directed by the CMV promoter. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson et al., Cell 37: 767 (1984). The fusion of the HA tag to the
target protein allows easy detection of the recombinant protein
with an antibody that recognizes the HA epitope.
[0201] The plasmid construction strategy is as follows. The PAPAI
cDNA of the deposited clone is amplified using primers that contain
convenient restriction sites, much as described above regarding the
construction of expression vectors for expression of PAPAI in E.
coli. To facilitate detection, purification and characterization of
the expressed PAPAI, one of the primers contains a hemagglutinin
tag ("HA tag") as described above.
[0202] Suitable primers include that following, which are used in
this example. The 5' primer has the sequence 5'CGC GGA TCC GCC ATC
ATG GAC ACA ATC TTC TTG 3' (SEQ ID NO:7) containing the underlined
BamH I restriction enzyme site, followed by 16 nucleotides
complementary to bp 67-84 of the antisense strand of the PAPAI
protein coding sequence set out in FIGS. 1A-1B (SEQ ID NO:1). For
the full length gene, the 3' primer has the full length sequence
CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA GGG ATT TGT CAC
TCT TCC (SEQ ID NO:9), containing the underlined Xba I restriction
site, an HA tag, and 18 nucleotides complementary to bp 1225-1242
of the sense strand of the PAPAI protein coding sequence set out in
FIGS. 1A-1B (SEQ ID NO:1).
[0203] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with BamH I and Xba I and then ligated. The ligation
mixture is transformed into E. coli strain SURE (available from
Stratagene Cloning Systems, 11099 North Torrey Pines Road, La
Jolla, Calif. 92037) the transformed culture is plated on
ampicillin media plates which then are incubated to allow growth of
ampicillin resistant colonies. Plasmid DNA is isolated from
resistant colonies and examined by restriction analysis and gel
sizing for the presence of the PAPAI-encoding fragment.
[0204] For expression of recombinant PAPAI, COS cells are
transfected with an expression vector, as described above, using
DEAE-DEXTRAN, as described, for instance, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989). Cells are incubated under
conditions for expression of PAPAI by the vector.
[0205] Expression of the PAPAI HA fusion protein is detected by
radiolabelling and immunoprecipitation, using methods described in,
for example Harlow et al., Antibodies. A Laboratory Manual, 2nd
Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1988). To this end, two days after transfection, the cells are
labeled by incubation in media containing .sup.35S-cysteine for 8
hours. The cells and the media are collected, and the cells are
washed and the lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5,
as described by Wilson et al. cited above. Proteins are
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
are analyzed by SDS-PAGE gels and autoradiography. An expression
product of the expected size is seen in the cell lysate, which is
not seen in negative controls.
Example 3
Cloning and Expression of the PAPA Protein in a Baculovirus
Expression System
[0206] The cDNA sequence encoding the soluble extracellular dornain
of PAPAI protein receptor protein in the deposited clone is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene:
[0207] The 5' primer has the sequence 5'CGC GGA TCC GCC ATC ATG GAC
ACA ATC TTC TTG 3' (SEQ ID NO:7) containing the underlined BamH I
restriction enzyme site followed by 18 bases (bp 67-84)
complementary to the antisense strand of the PAPAI protein coding
sequence of FIGS. 1A-1B (SEQ ID NO:1). Inserted into an expression
vector, as described below, the 5' end of the amplified fragment
encoding PAPAI protein receptor provides an efficient signal
peptide. An efficient signal for initiation of translation in
eukaryotic cells, as described by Kozak, M., J. Mol. Biol.
196:947-950 (1987), may be located, as appropriate, in the vector
portion of the construct.
[0208] For the full length gene, the 3' primer has the full length
sequence CGC GGT ACC TCA CTT CCT TTT ATC TCC CTG (SEQ ID NO:8),
containing the underlined Asp718 restriction followed by
nucleotides complementary to bp 1250-1267 of the sense strand of
the PAPAI protein set out in FIGS. 1A-1B (SEQ ID NO:1), and a stop
codon.
[0209] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("GENECLEAN.TM.," BIO 101 Inc.,
La Jolla, Calif.). The fragment then is digested with BamHI and
Asp718 and again is purified on a 1% agarose gel. This fragment is
designated herein F2.
[0210] The vector pA2 is used to express the PAPAI protein in the
baculovirus expression system, using standard methods, such as
those described in Summers et al., A Manual of Methods for
Baculovirus Vectors and Insect Cell Culture Procedures, Texas
Agricultural Experimental Station Bulletin No. 1555 (1987). This
expression vector contains the strong polyhedrin promoter of the
Autographa californica nuclear polyhedrosis virus (AcMNPV) followed
by convenient restriction sites. For an easy selection of
recombinant virus the beta-galactosidase gene from E. coli is
inserted in the same orientation as the polyhedrin promoter and is
followed by the polyadenylation signal of the polyhedrin gene. The
polyhedrin sequences are flanked at both sides by viral sequences
for cell mediated homologous recombination with wild-type viral DNA
to generate viable virus that express the cloned
polynucleotide.
[0211] Many other baculovirus vectors could be used in place of
pA2, such as pAc373, pVL941 and pAcIM1 provided, as those of skill
readily will appreciate, that construction provides appropriately
located signals for transcription, translation, trafficking and the
like, such as an in-frame AUG and a signal peptide, as required.
Such vectors are described, for example, in Luckow et al., Virology
170:31-39 (1989). Suitable vectors will be readily apparent to the
skilled artisan.
[0212] The plasmid is digested with the restriction enzymes BamH I
and Asp718 and then is dephosphorylated using calf intestinal
phosphatase, using routine procedures known in the art. The DNA is
then isolated from a 1% agarose gel using a commercially available
kit ("GENECLEAN.TM." BIO 101 Inc., La Jolla, Calif.). This vector
DNA is designated herein "V2".
[0213] Fragment F2 and the dephosphorylated plasmid V2 are ligated
together with T4 DNA ligase. E. coli HB 101 cells are transformed
with ligation mix and spread on culture plates. Bacteria are
identified that contain the plasmid with the human PAPAI protein
gene by digesting DNA from individual colonies using Bam HI and
Asp718 and then analyzing the digestion product by gel
electrophoresis. The sequence of the cloned fragment is confirmed
by DNA sequencing. This plasmid is designated herein as
pBacPAPAI.
[0214] 5 .mu.g of plasmid pBacPAPAI is co-transfected with 1.0
.mu.g of a commercially available linearized baculovirus DNA
("BACULOGOLD.TM. baculovirus DNA", Pharmingen, San Diego, Calif.),
using the lipofection method described by Felgner et al., Proc.
Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 .mu.g of BACULOGOLD.TM.
virus DNA and 5 .mu.g of the plasmid pBacPAPAI are mixed in a
sterile well of a microliter plate containing 50 .mu.l of serum
free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).
Afterwards, 10 .mu.l LIPOFECTIN.TM. plus 90 ll Grace's medium are
added, mixed and incubated for 15 minutes at room temperature. Then
the transfection mixture is added drop-wise to Sf9 insect cells
(ATTC.TM. CRL 1711) seeded in a 35 mm tissue culture plate with 1
ml Grace's medium without serum. The plate is rocked back and forth
to mix the newly added solution. The plate is then incubated for 5
hours at 27.degree. C. After 5 hours the transfection solution is
removed from the plate and 1 ml of Grace's insect medium
supplemented with 10% fetal calf serum is added. The plate is put
back into an incubator and cultivation is continued at 27.degree.
C. for four days.
[0215] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith, supra. An
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg,
Md.) is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
Md., at pages 9-10.
[0216] Four days after serial dilution, the virus is added to the
cells. After appropriate incubation, blue stained plaques are
picked with the tip of an Eppendorf pipette. The agar containing
the recombinant viruses is then resuspended in an Eppendorf tube
containing 200 .mu.l of Grace's medium. The agar is removed by a
brief centrifugation and the supernatant containing the recombinant
baculovirus is used to infect Sf9 cells seeded in 35 mm dishes.
Four days later the supernatants of these culture dishes are
harvested and then they are stored at 4.degree. C. A clone
containing properly inserted PAPAI protein receptor is identified
by DNA analysis including restriction mapping and sequencing. This
is designated herein as V-PAPAI.
[0217] Sf9 cells are grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells are infected with the recombinant
baculovirus V-PAPAI at a multiplicity of infection ("MOI") of about
2 (about 0.1 to about 3). Six hours later the medium is removed and
is replaced with SF900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Gaithersburg, Md.). 42
hours later, 5 .mu.Ci of .sup.35S methionine and 5 MCi .sup.35S
cysteine (available from Amersham) are added. The cells are further
incubated for 16 hours and then they are harvested by
centrifugation, lysed and the labeled proteins are visualized by
SDS-PAGE and autoradiography.
Example 4
Tissue Distribution of PAPAI Protein Expression
[0218] Northern blot analysis was carried out to examine the levels
of expression of PAPAI protein in human tissues, using methods
described by, among others, Sambrook et al, cited above. PolyA+ was
purchased from CLONTECH.TM. (1020 East Meadow Circle, Palo Alto,
Calif. 94303).
[0219] About 1 .mu.g of PolyA+ RNA was size resolved by
electrophoresis through a 1% agarose gel under strongly denaturing
conditions. RNA was blotted from the gel onto a nylon filter, and
the filter then was prepared for hybridization to a detectably
labeled polynucleotide probe.
[0220] As a probe to detect mRNA that encodes PAPAI protein, the
antisense strand of the coding region of the cDNA insert in the
deposited clone was labeled to a high specific activity. The cDNA
was labeled by primer extension, using the Prime-It kit, available
from Stratagene. The reaction was carried out using 50 ng of the
cDNA, following the standard reaction protocol as recommended by
the supplier. The labeled polynucleotide was purified away from
other labeled reaction components by column chromatography using a
Select-G-50 column, obtained from 5-Prime-3-Prime, Inc. of 5603
Arapahoe Road, Boulder, Colo. 80303.
[0221] The labeled probe was hybridized to the filter, at a
concentration of 1,000,000 cpm/ml, as described in Kreider et al.,
Molecular and Cellular Biology, September 1990, pp. 4846-4853.
Thereafter the probe solution was drained and the filter was washed
twice at room temperature and twice at 65.degree. C. with
0.1.times.SSC, 0.1% SDS. The filter then was then dried and exposed
to film at -70.degree. C. overnight with an intensifying
screen.
[0222] The Northern blot analysis showed maximal PAPAI transcript
levels in pancreas. Adipose also demonstrated a high abundant PAPAI
expression. No bands were present in other specimens analyzed,
including heart, brain, kidney, liver, lung, spleen, skeletal
muscle, esophagus, stomach, intestine, colon, uterus, placenta,
bladder, tonsil, thymus, appendix, lymph node, gall bladder,
prostate, testis and ovary.
[0223] Tissue expression of PAPAI appears to be limited. Although
large amounts of transcript were detected in pancreas and adipose
by Northern blot, no PAPAI transcript was detected in all other
tested tissues including liver and placenta which are the rich
sources for PAI-1 and PAI-2 respectively. PAPAI may function in a
tissue-specific fashion as part of an acute response to tissue
remodeling.
Example 5
Expression of PAPAI Protein in Normal and Cancerous Breast
Cells
[0224] The expression of PAPAI in human breast cancers was
investigated in a variety of human breast cancer cell lines. The
RNA from human breast cancer cells was prepared using the RNA
isolation kit RNAzol B (Tel-Test, Inc) based on manufacturer's
instructions. Equal aliquots of RNA were electrophoresed in a 1.2%
agarose gel containing formaldehyde and transferred to a nylon
membrane (Boehringer Mannheim). The membrane was pre-hybridized
with EXPRESSHYB.TM. hybridization solution (CLONTECH.TM., Inc.) at
68.quadrature.C for 30 min. The hybridization was carried out in
the same solution with .sup.32P_labeled PAPAI probe
(1.5.times.10.sup.6 cpm/ml) for 1 hour at 68.quadrature.C. The
membrane was then rinsed in 2.times.SSC containing 0.05% SDS three
times for 30 min at room temperature followed by two washes with
0.1.times.SSC containing 0.1% SDS for 40 min at 55.quadrature.C.
The full-length PAPAI cDNA was isolated from the Bluescript vector
followed by EcoRI and XhoI digestion and used for preparation of a
cDNA probe.
[0225] The Northern blot analysis failed to detect the PAPAI
transcript in all tested breast cancer cell lines. The inability to
pick up the PAPAI mRNA in breast cancer cell lines by Northern blot
indicates that the PAPAI gene may be only expressed in
myoepithelial cells.
[0226] In order to localize the cellular source of PAPAI expression
and to assess the biological relevance of PAPAI expression in
breast cancer's progression, in situ hybridization was performed on
the fixed sections from a variety of different human breast
specimens. In this example, two aspects of PAPAI expression were
examined, including the tissue localization (stromal versus
epithelial) and the potential correlation of the loss of PAPAI
expression and breast cancer malignant phenotype. A 1176 bp PAPAI
cDNA was inserted into pBluescript SK at unique EcoRI (5') and XhoI
(3') sites. The digoxigenin-labeled antisense probe was generated
by an EcoRI cut of PAPAI cDNA plasmid and followed by T7 RNA
polymerase (Boehringer Mannheim). Deparaffinized and rehydrated
tissue sections were treated with 0.2 M HCl at room temperature for
20 min and de-proteinized in 0.2 mg/ml proteinaseK solution at
37.degree. C. for 30 min. The slides were then acetylated in 0.4%
acetic anhydride in 0.1M triethanolanine for 10 min and dehydrated
in ethanol. Prehybridization was carry out in hybridization buffer
(0.3M NaCl, 0.02 Sodium Acetate, 5 mM EDTA, pH 5.0), 50% formamide,
1.times. Denhardt's, 10% dextran sulfate and 1 mg/ml tRNA over
night in a humidity chamber at 50.degree. C. Thereafter, the
sections were hybridized with 500 ng/ml DIG-labeled RNA probe in
the same components as those in the prehybridization step at
50.degree. C. for 18 h. After hybridization, the slides were then
washed with 2.times.SSC, 0.2.times.SSC, 0.1.times.SSC separately at
42.degree. C. for 20 min and treated with Rnase A in 0.1.times.SSC
at 37.degree. C. for 30 min. Digoxigenin detection was performed
with mouse antidigoxigenin antibodies (Boehringer Mannheim)
followed by incubation with biotin-conjugated secondary rabbit
antimouse antibodies (DAKO). The colorimetric detections were
performed with a standard indirect streptavidin-biotin
immunoreaction method using the Universal LSAB Kit (DAKO) according
to the manufacture's instructions.
[0227] There was a strongly positive PAPAI hybridization in the
myoepithelial cells surrounding the normal mammary glands, benign
hyperplasias, and benign fibroadenomas. The expression of PAPAI
mRNA was detectable in the myoepithelial cells in all four
reduction mammoplasty specimens and in three benign lesions. In
contrast, expression of PAPAI was absent in five out of five cases
of infiltrating breast carcinomas. No PAPAI expression can be
detected in both normal and malignant mammary epithelial cells and
in stromal fibroblasts. In all cases a strong PAPAI transcript was
found in the endothelial cells of small vessels. These in situ
hybridization results are consistent with the Northern blot
analysis which showed no PAPAI expression in breast cancer
cells.
[0228] The loss of PAPAI expression in the malignant breast
carcinomas may be due to the loss of putative PAPAI-producing
myoepithelial cells during the malignant progression. It is
intriguing that these data are different from the previous studies
that have linked excessive PAI-1 and PAI-2 expression to breast
cancers as compared to normal breast (Duggan, C. et al., Br. J.
Cancer 76(5):622-627 (1997); Brunner, N. et al., Cancer Treat. Res.
71:299-309 (1994); Duffy, M. et al., Cancer 62, 531-533 (1988);
Duggan, C. et al., Int. J. Cancer 61:597-600 (1995); Schmitt, M. et
al., Br. J. Cancer 76(3):306-311 (1997); Bianchi, E. et al., Int.
J. Cancer 60:597-603 (1995); and Bouchet, C. et al., Br. J. Cancer
69:398-405 (1994)). Both in situ hybridization and
immunohistochemical staining have demonstrated a strong PAI-1
expression in the stroma surrounding breast carcinomas or at tumor
margins (Bianchi, E. et al., Int. J. Cancer 60:597-603 (1995)). In
particular, it was extensively demonstrated that the clinical
outcome of breast cancer is reversely related to the levels of
PAI-1 expression (Liu, G. et al., Int. J. Cancer 60:501-506
(1995)). It is not easy to understand why the elevated tumor tissue
content of PAI-1 indicates a poor prognosis for the breast cancer
patients. One explanation is that, the increased expression of
PAI-1 may be reciprocally related to the increased expression of
u-PA and ohter proteinases during the tumor-mediated degradation of
extracellular matrix. Therefore, this elevated levels of PAI-1 in
the stroma adjunct to the invasive breast carcinomas may represent
one of the subsequent acute host responses to the remodeling
stimuli and try to balance the local tissue degradation, but not as
a causative factor. Alternatively, the high level expression of
PAI-1 in the breast cancer may favor the proposed co-expression
model that uPA and PAI-1 have to be present in the tumor in order
to achieve focalized and optimal uPA-R-mediated proteolysis and
invasiveness (Dickinson, J.-L. et al., J. Biol. Chem.
270:27894-27904 (1995)).
[0229] PAI-2 most probably is acting on tumor cells in a different
way than PAI-1. In contrast to PAI-1, although the PAI-2 expression
is also increased in breast cancers relative to normal breast,
PAI-2 expression is detected predominantly in the malignant breast
cancer cells and high levels of PAI-2 has been proposed as a
favourable prognostic marker in breast cancer (Duggan, C. et al.,
Br. J. Cancer 76(5):622-627 (1997)). Since PAI-2 is mainly found as
an intracellular protein, this raises a possibility as to its
physicological role on protection of the cells from some harmful
effects of an intracellular proteinases (Schmitt, M. et al.,
Thromb. Haemost. 78(1):285-296 (1997)). PAI-2 is an important
factor in cytoprotection of cell death in TNF.alpha.-induced
process through inhibition of the proteinases involved in
TNF.alpha.-mediated apoptosis (Dickinson, J.-L. et al., J. Biol.
Chem. 270:27894-27904 (1995)). This PAI-2-mediated cytoprotective
role resembles the antiapoptotic action of bcl-2 gene which is in
close proximity of the PAI-2 gene on chromosome 18 (Dickinson,
J.-L. et al., J. Biol. Chem. 270:27894-27904 (1995); Bachmann, F.,
Thromb. and Haemostas. 74(1):172-179 (1995); and Sternlicht, M.-D.
& Barsky, S.-H, Med. Hypoth. 48:37-46 (1997)). In this regard,
the increased expression of PAI-2 in breast cancer cells may be in
part responsible for the less apoptotic phenotype of the malignant
cancer cells. On the other hand, a secreted glycoslated form of
PAI-2 is also present (Bachmann, F., Thromb. and Haemostas.
74(1):172-179 (1995)) and may contribute its anti-invasive and
anti-metastatic effect through inhibition of PA.
[0230] It is interesting to note that PAPAI expression is seen
exclusively in the myoepithelial cells which are lost during the
breast cancer malignant progression. Myoepithelial cells, normally
surrounding ducts of glandular organs such as breast, contribute to
the synthesis of a surrounding basement membrane and exert
important paracine effects on epithelial mitogenesis and
morphogenesis (Sternlicht, M.-D. & Barsky, S.-H Med. Hypoth.
48:37-46 (1997)). In normal or non-invasive benign breast,
cell-stromal contact is mediated by myoepithelial cells which
secrete relatively low levels of matrix-degrading proteinases but
relatively high levels of maspin and various other anti-invasive
proteinase inhibitors (Stemlicht, M.-D., Safarians, S., Rivera,
S.-P. & Barsky, S.-H. Lab Invest. 74 (4), 781-796 (1996)).
Myoepithelial cells can also induce differentiation of breast
cancer cells (Bani, D. et al., Br. J. Cancer 70(5):900-904 (1994))
and inhibit tumor cell invasion in vitro (Stemlicht, M.-D. &
Barsky, S.-H Med. Hypoth. 48:37-46 (1997)). The exclusive
expression of PAPAI in myoepithelial cells of normal or benign
manmary gland may represent one of the major anti-invasive
proteinase inhibitors mediated by the host defensive myoepithelial
layer in preventing breast cancer malignant progression. In this
regard, the expression of PAPAI in myoepithelial cells would create
a micro-environment in the epithelial-stromal interface where the
inhibitory effect of PAPAI prevents the excessive proteolytic
actions and preserve the epithelial-stromal structure integrity. It
has been recognized that an intact myoepithelial layer, like an
intact extracellular basement, can distinguish benign epithelial
proliferations and in situ carcinomas from invasive disease. The
exclusive expression of PAPAI in the myoepithelial cells may play a
role as an anti-invasive and anti-metastatic phenotype in the
myoepithelial layer.
Example 6
Affect of PAPAI on Breast Cancer Progression
[0231] Reagents. Restriction enzymes, T7 polymerase, random primer
DNA labeling kit, and digoxigenin-labeled nucleotides were obtained
from Boehringer Mannhem, Indianapolis. .sup.32P-dATP was purchased
from Amersham.
[0232] In vitro assay for cell growth. Exponentially growing
cultures of different MDA-MB-435 clones were detached with trypsin,
and the trypsin was neutralized with DMEM-10% serum. Cells were
counted, diluted, and seeded in triplicate at 3,000 cells per well
(24-well plate) in 1 ml DMEM-5% serum. Cell growth was measured
using CellTiter 96.TM. Aqueous Non-Radioactive cell proliferation
Assay Kit (Promega).
[0233] In vitro invasion assay. The modified Boyden chamber
invasion assay was performed as previously described (Wang, M. et
al., Oncogene 14 (23):2767-2774 (1997)).
[0234] Tumor growth, lymph node and lung nietastasis, and
microvessel counts in athymic nude mice. A nude mouse mammary fat
pad tumorigenic and metastatic assay was performed as previously
described (Shi, Y.-E., Cancer Res. 57:759-764 (1997) and Shi, Y.-E.
et al., Cancer Res. 57(15):3084-3091 (1997) and Wang, M. et al.,
Oncogene 14(23):2767-2774 (1997)). Microvessel counts of primary
tumors was analyzed as we previously described for TIMP-4
transfected MDA-MB-435 tumor model (Wang, M. et al., Oncogene
14(23):2767-2774 (1997)).
[0235] Statistical Analysis. Values were expressed as
means.+-.standard errors (SEs). Comparisons were made using the
two-tailed Student's t-test. Where appropriate, the chi-squared
test was used to compare proportions.
[0236] Transfection and selection of PAPAI positive clones. Since
the expression of PAPAI in the myoepithelial cells may contribute
the roles of myoepithelial layer as paracrine cellular suppression
of invasion, it was tested whether we can inhibit breast cancer
invasion and metastasis by transfection of PAPAI into metastatic
breast cancer cells. In order to select a suitable breast cancer
cell line for PAPAI gene transfection, a panel of breast cancer
cell lines as well as normal mammary epithelial cells were screened
for analysis of PAPAI expression. Northern blot analysis failed to
detect the PAPAI transcript in MCF-7, T47D, MDAMB-231, MDAMB-435,
MDAMB-436, ZR 75-1, Hs578t and BT549 breast cancer cell lines and
NME4144 and NME4244 normal mammary epithelial cells. The inability
to pick up the PAPAI mRNA in both normal and malignant breast
epithelial cells by Northern blot supports the in situ
hybridization data indicating the myoepithelial expression of the
PAPAI.
[0237] MDA-MB-435 cell line was selected as recipient for PAPAI
mediated gene transfection because: 1) it lacks detectable PAPAI
transcript; and 2) it is relatively highly tumorigenic and
metastatic in nude mice. Human PAPAI cDNA was subcloned into the
pCI-neo mammalian expression vector (Promega) downstream of the
human cytomegalovirus promoter and enhancer to generate the pCNPAI
expression vector. 40 .mu.g pCNPAI or the control vector pCI-neo
were used for transfections. 1.times.10.sup.6 MDA-MB-435 human
breast cancer cells were plated 24 h prior to transfection on
100-mm dishes and then incubated with IMEM supplemented with 10%
FBS, 100 IU/ml penicillin G, 100 mg/ml streptomycin and 2 mM/ml
L-glutamine (GIBCO, BRL). Transfection was carried out using the
calcium phosphate coprecipitation method. DNA was removed 16 h
later by replacing the incubation medium. 24 h later, the cells
were subcultured to five 100-mm dishes containing IMEM supplemented
with 10% FBS, 100 IU/ml penicillin G, 100 mg/ml streptomycin, 2
mM/ml L-glutamine and 0.8 mg/ml G418 (Geneticin, GIBCO). After
colonies of about 104 cells had grown, 30 G418-resistant individual
clones were picked and subcloned. Clones were initially screened by
in situ hybridization on slides with a specific PAPAI antisense
probe, and the positive clones were subjected to Northern blot
analysis. MDA-MB435 subclones transfected with PAPAI cDNA were
designated PAPAI-435, and MDA-MB-435 subclones transfected with
pCI-neo were designated neo-435. Five PAI-435 clones were picked up
by in situ hybridization. All five selected PAI-435 clones,
PAI-435-1, PAI-435-5, PAI-435-6, PAI-435-10, and PAI-435-11
expressed PAI mRNA transcripts. In contrast, none of the parental
MDA-MB-435 cells or cells transfected with plasmid vector alone
produced any detectable PAPAI transcripts.
[0238] Expression of plasminogen activator inhibitor activity. The
anti-tPA activity of PAPAI transfected clones was characterized.
Conditioned media from two PAI-435 clones, one control clone and
parental MDA-MB-435 cells were collected, concentrated, and
analyzed for plasminogen activator inhibitory activity. Although
the basal level PAI-like activities were detected in PAPAI negative
clones, the anti-tPA activities in two PAPAI positive clones were
three times higher than that of PAPAI negative clones. These
results indicate that the PAPAI transfected clones secreted a
functional PAPAI protein.
[0239] In vitro growth of PAPAI-435 cells. To determine whether
PAPAI expression affects the growth of MDA-MB-435 cells, the growth
rates of PAPAI-435-1 and PAPAI-435-10 cells were compared to that
of neo-435-2 and neo-435-4 cells in the monolayer culture. No
significant differences in growth rate were observed among PAPAI
positive and PAPAI negative cells.
[0240] Effect of PAPAI transfection on tumorigenicity. To study the
effect of PAPAI expression on tumorigenicity, picked two PAPAI
positive clones were tested: PAPAI-435-1 and PAPAI-435-10; and two
PAPAI negative cells: neo-435-2 and neo-435-4. A pilot study was
done and the data are summarized in Table 2. After a lag phase of
7-10 days, all the 20 injections in the mice given implants of
PAPAI negative cells developed tumors. In contrast, only 10
injections in the mice given implants of PAPAI positive cells
developed tumors. Starting at about 27 days after inoculation,
great level of tumor necrosis was observed in tumors derived from
neo-435-1 and neo-435-10 cells. The same breast cancer cells
transfected with PAPAI, however, were significantly inhibited in
their tumor growth; and either no or low level of tumor necrosis
was observed. The size of PAPAI-435-10 tumors was only 23% of that
in parental neo-435-1 tumors and 21% of that in neo-435-10 tumors.
In addition, the tumor incidence was also greatly decreased. With
10 injections, only two implants developed tumors. The tumor growth
of PAPAI-435-1 cells was also significantly reduced, with 54% and
52% inhibition of tumor size observed as compared to neo-435-2 and
neo-435-15 tumors, respectively. TABLE-US-00002 TABLE 2 Effects of
PAPAI expression on tumor sizes and tumor incidence. Tumor vol
(mm.sup.3) Tumor incidence Treatment Group of primary size
Tumor/Total 435-neo-2 366.81 .+-. 75.26 10/10 (100%) 435-neo-4
387.38 .+-. 91.72 10/10 (100%) PAPAI-435-1 199.54 .+-. 32.88 8/10
(80%) PAPAI-435-10 83.13 .+-. 8.41 2/10 (20%)
Discussion
[0241] In this example, the biological relevance of PAPAI in human
breast cancer progression was characterized. Amino acid sequence
analysis indicate that PAPAI shares considerable sequence
similarity with members of the serpin family, including PAI-1 and
PAI-2. Like PAI-1, a cleavable hydrophobic signal sequence is
identified for PAPAI, indicating its secretion. PAPAI expressed in
MDA-MB-435 human breast cancer cells. As expected, the resulting
recombinant PAPAI protein possesses an inhibitory activity on PA
and is secreted extracellularly, thus confirming that the novel
protein is a new member of the PAI family.
[0242] Transfection of the MDA-MB-435 cells with a PAPAI cDNA leads
to increased expression of the PAPAI transcript and anti-tPA
activity when compared to parental cell line and control cells. The
reduced in vitro invasiveness of PAPAI-435 clones compared to
control cells suggests that the production of PAPAI altered the
invasive potential of breast cancer cells in this experimental
model system. These results are consistent with the previous
reports on the inhibition of the invasion by PAI-1 (Soff, G.-A. et
al., J. Clin. Invest. 96:2593-2600 (1995)) and PAI-2 (37-38). In
our nude mouse model of mammary tumor, overexpression of PAPAI
resulted in several phenotypic changes: (a) there was a significant
reduction in incidence and size of primary tumors; (b)
tumor-associated angiogenesis was inhibited as evidenced by the
reducted microvessel density in the tumor; (c) the number of
microscopic metastatic lesions in the lung and lymph node was
reduced. The PAPAI-mediated in vivo tumor growth inhibition is
somewhat conflicting to the in vitro similar growth rates of
PAPAI-positive clones compared to PAPAI-negative clones. The slower
in vivo growth of PAPAI435 tumors may be explained, in part, by
PAPAI-mediated inhibition of tumor angiogenesis. Angiogenic
regulatory factors have been found to modulate the growth of human
breast cancers in several orthotopic xenograft models. We have
recently demonstrated that transfection of MDA-MB-435 cells with an
angiogenic factor Scatter Factor (SF) increased tumor growth and
angiogenesis (Mignati, P., Tsuboi, R., Robbins, E. & Rifkin,
D.-B. J. Cell Biol. 108, 671-682 (1989)); in contrast,
overexpression of tissue inhibitor of metalloproteinase 4 (TIMP-4)
in MDA-MB-435 cells inhibited primary tumor growth, metastasis, and
tumor angiogenesis (Wang, M. et al., Oncogene 14 (23):2767-2774
(1997)). The data in this example indicate that despite the lack of
growth inhibition of PAPAI on breast cancer cells, PAPAI
significantly inhibits tumor growth and metastasis presumably due
to its anti-angiogenic activity. In fact, uPA stimulates components
of angiogenesis including chemotaxis, proteolytic matrix
degradation, and the release of basic fibroblast growth factor from
its storage in the basement membrane (Yasunaga, C. et al., Lab.
Invest. 61:689-704 (1989); Mignati, P. et al., J. Cell Biol.
108:671-682 (1989); Saksela, O. & Rifkin, D.-B. J. Cell Biol.
110: 767-775 (1990); and Flaumenhaft, R. et al., J. Cell Physiol.
140:75-81 (1989)). Furthermore, both PAI-1 (Soff, G.-A. et al., J.
Clin. Invest. 96:2593-2600 (1995)) and PAI-2 have been demonstrated
to have an anti-angiogenic activity presumably through inhibition
of uPA expressed by endothelial cells in newly forming capillary
sprouts.
[0243] The magnitude of the tumor-suppressing activity of PAPAI on
human breast cancer is greater than that observed previously for
metalloproteinase inhibitor TIMP-4 (Wang, M. et al., Oncogene
14(23):2767-2774 (1997)) and comparable to that observed for tumor
suppressor Rb and p53 (Wang, N.-P. et al., Oncogene 8:279-288
(1993)). The exclusive expression of PAPAI in the myoepithelial
cells of normal and benign breast and the inhibition of breast
tumor growth and metastasis by PAPAI expression suggest that PAPAI
is one of the local myoepithelial-related paracrine factors that
preserve the normal epithelial-stromal integrity and prevent the
malignant progression from benign or in situ to the metastatic
phenotype.
[0244] The entire disclosure of each document cited in this
application is hereby incorporated herein by reference.
Sequence CWU 1
1
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